Compositions and methods for bioprinting of energy-responsive bioinks

Abstract

Disclosed are methods and compositions to enable bioprinting of energy-responsive bioinks. The embodiments of the present disclosure relate to ultrasound-controlled gene delivery to a target cell in a three-dimensional bioprinted cell construct. The compositions include, for example, a hydrogel scaffold with a plurality of filament structures printed on a substrate; and a collagen cast gel that encases the hydrogel scaffold; wherein the compositions are configured to receive a population of microparticle-coupled cargo for controlled delivery to one or more target cells by application of ultrasound energy. Also disclosed are methods of creating transgenic target cells using the disclosed compositions.

Description

COMPOSITIONS AND METHODS FOR BIOPRINTING OF ENERGY-RESPONSIVE BIOINKS CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 707,180, filed October 14, 2024, the entire contents of which are hereby incorporated by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under R35GM151057 awarded by the National Institutes of Health and under 2339254 awarded by the National Science Foundation. The government has certain rights in the invention. FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to compositions and methods to enable bioprinting of energy-responsive bioinks. Embodiments provided herein particularly relate to ultrasound- controlled delivery of microparticle-coupled cargo in three-dimensional bioprinted cell constructs and transgene expression for improvements in the treatment or modeling of diseases. BACKGROUND OF THE DISCLOSURE

[0004] Coaxial 3D bioprinting has been established as a versatile fabrication method for tissue constructs due to its ability to produce complex tissue-like architectures containing multiple cell types and to mimic biophysical parameters of the matrix microenvironment. Compared to 2D culture, 3D culture better recapitulates in-vivo properties such as cell-cell and cell-matrix interactions, protein expression, and enzyme activity.143D model systems are also more amenable to cellular imaging than in-vivo models.1,44Three-dimensional bioprinting provides advantages for creating cell-laden architectures, such as spatial control over matrix properties32, biochemical factors20, and cell type.39

[0005] Coaxial bioprinting, which utilizes a nozzle with multiple concentric orifices, has garnered significant interest as an extrusion-based bioprinting method, as it allows for precisely controlled simultaneous bioprinting of multiple bioinks to form complex constructs.30By having a core material and outer sheath material that can each be printed with different parameters, coaxial bioprinting enables the co-printing of hydrogel bioinks with different properties, such as those with complementary mechanical or biochemical features. The concentric nozzle design alsoallows for single-step crosslinking by extrusion of the bioink from the core and the crosslinking agent from the outer sheath. Conversely, the crosslinker can be extruded from the core with bioink in the sheath to form hollow fibers.40Coaxial bioprinting can also incorporate sacrificial materials in a single printing step, such as a sacrificial core to form a hollow tubular structure.25,30,40These features make coaxial bioprinting an important method for creating 3D tissue constructs, as it enables the production of more physiologically relevant tissue architectures, such as vasculature21,51,52,58as well as zonal cartilage organization and intestinal villus structures.24,29,30

[0006] The ability to genetically manipulate cells within coaxially bioprinted tissue constructs is an essential tool for modeling dynamic changes in cell signaling and enables the perturbation of cell behaviors. This has utility for stimulating intercellular communication, growth factor release, and stem cell differentiation in tissue constructs18to direct cell function in the 3D microenvironment, and can also be used to model disease states. Spatiotemporal control over gene delivery is critical to allow cell structures within constructs to self-assemble and mature, forming cell-cell connections and signaling interactions before initiating genetic changes in selected cells or tissue regions. This is an important order of events to model genetic changes within fully formed tissue structures. Remote genetic manipulation also enables the ability to transform a small subset of user-defined cells within the established tissue construct which is different from transforming all the cells all at once. This allows researchers to study how these transformed cells interact with the normal cells around them, in a biomimetic tissue environment. An important application for controlled genetic manipulation in 3D constructs includes disease modeling including cancer development, which involves genetic and epigenetic changes45in a subset of cells within a mature tissue that has established connections and intercellular signaling.

[0007] However, achieving spatial and temporal control over genetic manipulation of cells within coaxially-bioprinted constructs is a challenge because scaffold matrices hinder the diffusion of traditional transfection vectors, making it difficult to control their localization. Current methods for gene delivery used in 2D culture, such as commonly used viral transduction, lack the ability to both spatially and temporally control the genetic manipulation of cells within 3D constructs.17Additionally, biomaterial scaffolds present challenges for viral transduction, as thick constructs and crosslinked filaments can hinder virus diffusion, which can result in uneven gene delivery.33Chemical transfection methods allow for cross-membrane DNA delivery, but lack spatiotemporal control of genetic manipulation within the 3D tissue construct.70Gene activated matrices (GAM) are a platform for localized and sustained gene delivery in biofabricated 3D constructs, andhave shown potential for directing gene expression in 3D scaffolds.60,66In these systems, nucleic acids are incorporated into the construct matrices and the GAM platform facilitates the sustained release of nucleic acids to embedded cells. GAM-based DNA delivery has also been shown to be effective at delivering DNA to cells in-vivo after implantation into tissue.41 31However, These GAM-based strategies rely on passive diffusion or cellular motility for the incorporated DNA to interact with cells which does not allow for precise control over when and where the DNA is delivered. Therefore, current strategies are limited in their ability to create a relevant 3D architecture containing multiple cell types and lack the function of spatial and temporal gene delivery. There remains an unmet need for developing new platforms and methods that may allow genetic manipulation in tissue constructs with precise control. SUMMARY OF THE DISCLOSURE

[0008] Embodiments provide for a composition comprising a hydrogel scaffold with a plurality of filament structures printed on a substrate; and a collagen cast gel that encases the plurality of filament structures of the hydrogel scaffold; wherein the composition is configured to receive a population of microparticle-coupled cargo for controlled delivery to one or more target cells by application of ultrasound energy. In some embodiments, the population of microparticle-coupled cargo comprises a population of gas-filled microbubbles coupled to a substance selected from the group of nucleic acid, peptide, polypeptide, amino acid, protein, and drug, or any combinations thereof.

[0009] Embodiments also provide for a composition comprising a hydrogel scaffold with a plurality of filament structures; a collagen cast gel encasing the hydrogel scaffold; a population of mammalian cells incorporated into the hydrogel scaffold; and a plurality of microbubbles dispersed throughout the filament structures of the hydrogel scaffold, wherein the microbubbles are attached to one or more nucleic acid molecules to be expressed in the mammalian cells. In some embodiments, the hydrogel scaffold comprises alginate. In some embodiments, the composition is configured to be responsive to an ultrasonic frequency ranging from about 0.5 MHz to about 15 MHz.

[0010] Embodiments also provide for a method of creating a transgenic target cell, the method comprising the steps of: (a) providing a hydrogel scaffold with a plurality of filament structures encased in a collagen cast gel, wherein the filament structures comprise a plurality of target cells and a population of ultrasound-responsive microbubbles stably bound to one or more nucleic acid molecules; (b) applying ultrasound to a focused region of the hydrogel scaffold with sufficient energy to cavitate the microbubbles causing an uptake of the one or more nucleic acidmolecules by at least one target cell from the plurality of target cells present in proximity to the cavitated microparticles within the focused region of the hydrogel scaffold; and (c) imaging the ultrasound-exposed hydrogel scaffold to visualize the at least one target cell that comprises the one or more nucleic acid molecules; wherein the at least one target cell expressing the one or more nucleic acid molecules is the transgenic target cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Some of the drawings submitted herein may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserve the right to present color images of the drawings in later proceedings.

[0012] Figure 1. 3D bioprinting of microbubble gene delivery vehicles. Schematic depicting coaxial bioprinting of a 3D construct containing cells and DNA-coupled ultrasound-responsive microbubbles. The construct is printed with a coaxial needle where the needle core contains sodium alginate bioink precursor solution, microbubble gene delivery vehicles, and cells. The sheath compartment contains calcium crosslinking agent thereby enabling printing of microbubble-containing crosslinked hydrogel filaments. Post-printing, focused ultrasound is applied to user defined areas of the construct resulting in spatiotemporally controlled regions of gene delivery.

[0013] Figures 2A-2F. Ultrasound-responsive microbubble gene delivery vehicles. Figs. 2A- 2C show representative microscopy images of DNA-coupled ultrasound-responsive microbubbles in brightfield (Fig.2A), fluorescence (plasmid DNA visualized via YOYO-1 stain) (Fig. 2B), and brightfield / fluorescence overlay (plasmid DNA visualized via YOYO-1 stain) (Fig. 2C). Fig. 2D shows representative size distribution of DNA-coupled ultrasound-responsive microbubbles. Figs.2E-2F show high magnification microscopy image of DNA-coupled ultrasound-responsive microbubble in brightfield (Fig. 2E), showing the dark microbubble and characteristic ring distortions surrounding it due to the lensing effect of the gas bubble core, and fluorescence (plasmid DNA visualized via YOYO-1 stain) (Fig. 2F).

[0014] Figures 3A-3C. Coaxial bioprinting of microbubble gene delivery vehicles. Figs. 3A-3C show representative macroscale and microscopy images of coaxially-bioprinted alginate at 2% w / v (Fig. 3A), 4% w / v (Fig. 3B), 6% w / v (Fig. 3C) with and without ultrasound-responsive microbubble (μB) gene delivery vehicles. Black arrows indicate filament boundaries.

[0015] Figures 4A-4C. Stability of coaxially-printed microbubble gene delivery vehicles. Fig. 4A shows representative microscopy images of coaxially-bioprinted HEK293T-laden 4% w / v alginate filaments containing microbubbles from 0-48 hr, in samples kept incubated at 37°C (toprow) and at room temperature (RT, bottom row). Fig. 4B shows quantification of microbubble stability over time in 4% w / v alginate in samples incubated at 37°C and at room temperature. Fig. 4C shows representative fluorescent image of bioprinted filament containing microbubbles and Hoechst stained HEK293T cells.

[0016] Figures 5A-5D. Effect of microbubble concentration on the size of the ultrasound induced activation zone in 4% w / v alginate bioprinted constructs. Figs. 5A-5C show brightfield images of 4% w / v alginate pre- and post-ultrasound (US) exposure containing 1.56x109μB / mL (Fig. 5A), 2.34x109μB / mL (Fig. 5B), and 3.51x109μB / mL (Fig. 5C). Fig. 5D shows quantified size of microbubble activation zone in 4% alginate with varying microbubble concentrations (** p<0.01, N=3, one-way ANOVA, Tukey’s multiple comparisons test, error bars denote standard deviation).

[0017] Figure 6. Viability of bioprinted cells with ultrasound-responsive microbubbles in alginate bioinks. The graph shows viability of HEK293T cells printed in 2%, 4%, and 6% w / v alginate at 0 hr and 48 hr post-printing (N=3, error bars denote standard deviation).

[0018] Figures 7A-7C. Effect of varying the number of ultrasound pulses on ultrasound- controlled transfection of HEK293T cells in coaxially-bioprinted constructs. Fig. 7A shows coaxially-bioprinted HEK293T-laden 4% alginate bioink containing GFP-coupled microbubbles before ultrasound, 0 hr post-ultrasound, and 48 hr post-ultrasound with varying ultrasound exposure (10, 40, or 80 pulses). Fig. 7B shows number of transfected cells in bioprinted constructs at 48 hr post-ultrasound for varying numbers of ultrasound pulses (* p<0.05, ** p<0.01, one-way ANOVA, Tukey’s multiple comparisons test, error bars denote standard deviation). Fig. 7C shows diameter of zone containing transfected cells at 48 hr post-ultrasound for varying numbers of ultrasound pulses (one-way ANOVA, Tukey’s multiple comparisons test, error bars denote standard deviation).

[0019] Figures 8A-8C. Effect of increasing microbubble concentration on ultrasound- controlled HEK293T cellular transfection in coaxially-bioprinted constructs. Fig. 8A shows increasing concentrations of GFP-coupled microbubbles in coaxially-bioprinted HEK293T-laden 4% alginate bioink, before ultrasound, 0 hr post-ultrasound, and 48 hr post-ultrasound. Fig. 8B shows number of transfected cells in bioprinted constructs at 48 hr post-ultrasound for varying microbubble concentrations (* p<0.05, one-way ANOVA, Tukey’s multiple comparisons test, error bars denote standard deviation). Fig. 8C shows diameter of zone containing transfected cells at 48 hr post-ultrasound for varying microbubble concentrations (** p<0.01, one-way ANOVA, Tukey’s multiple comparisons test, error bars denote standard deviation).

[0020] Figures 9A-9C. Effect of varying the number of ultrasound pulses on ultrasound- controlled osteoblast cellular transfection in coaxially-bioprinted constructs. Fig.9A shows coaxially-bioprinted osteoblast-laden (hFOB 1.19) 4% alginate bioink containing GFP-coupled microbubbles before ultrasound, 0 hr post-ultrasound, and 48 hr post-ultrasound with varying ultrasound exposure (10, 40, or 80 pulses). Fig. 9B shows number of transfected cells in bioprinted constructs at 48 hr post-ultrasound for varying numbers of ultrasound pulses (* p<0.05, **** p<0.0001, one-way ANOVA, Tukey’s multiple comparisons test, error bars denote standard deviation). Fig. 9C shows diameter of zone containing transfected cells at 48 hr post- ultrasound for varying numbers of ultrasound pulses (one-way ANOVA, Tukey’s multiple comparisons test, error bars denote standard deviation).

[0021] Figure 10. Representative images of coaxially-bioprinted HEK293T-laden 4% alginate bioink filament containing GFP-plasmid loaded microbubbles (at 2.34 x 109μB / mL) that was not exposed to ultrasound. Filament shown via brightfield microscopy (left), and fluorescence microscopy at 48 hr post-printing (middle, right).

[0022] Figure 11. Representative images of coaxially-bioprinted HEK293T-laden 4% alginate bioink filament containing no microbubbles and no plasmid DNA, exposed to 40 ultrasound pulses. Filament shown in brightfield before and after ultrasound exposure, and via fluorescence microscopy at 48 hr post-ultrasound exposure.

[0023] Figure 12. Representative images of coaxially-bioprinted HEK293T-laden 4% alginate bioink filament containing microbubbles with no plasmid DNA (at 2.34 x 109μB / mL), exposed to 40 ultrasound pulses. Filament shown in brightfield before and after ultrasound exposure, and via fluorescence microscopy at 48 hr post-ultrasound exposure.

[0024] Figure 13. Expanded view of full transfection zone of coaxially-bioprinted HEK293T- laden 4% alginate filament printed with 7.81 x 108μB / mL and exposed to 40 ultrasound pulses. Expanded view obtained from stitched post-transfection fluorescence images from the sample appearing in Figure 8A.

[0025] Figure 14. Representative images of coaxially-bioprinted osteoblast-laden (hFOB 1.19) 4% RGD-alginate bioink filament containing GFP-plasmid loaded microbubbles (at 2.34 x 109μB / mL) that was not exposed to ultrasound. Filament shown via brightfield microscopy (left), and fluorescence microscopy at 48 hr post-printing (middle, right).

[0026] Figure 15. Representative images of coaxially-bioprinted osteoblast-laden (hFOB 1.19) 4% RGD-alginate bioink filament containing no microbubbles and no plasmid DNA, exposed to 40 ultrasound pulses. Filament shown in brightfield before and after ultrasound exposure, and via fluorescence microscopy at 48 hr post-ultrasound exposure.

[0027] Figure 16. Schematic of the ultrasound induced sonoporation and localized delivery of DNA to cells within a 3D hydrogel construct. The lipid-coated microbubbles are positively charged and can electrostatically bind negatively charged plasmid DNA on the surface. The microbubbles are mixed into the fluid hydrogel precursor solution, along with the cells, allowing for even distribution throughout the construct once polymerized. Focused ultrasound activates the microbubbles causing inertial cavitation which delivers DNA to nearby cells through sonoporation. This only occurs in the ultrasound focal zone enabling both temporal and spatial control over cell genetic expression within the scaffold. The ultrasound is noninvasive and leaves intact any self-organized cellular structures formed before ultrasound exposure.

[0028] Figures 17A-17B. Schematic of final bioprinting construct embedded in cast collagen gel. Fig. 17A shows the bioprint, comprising filaments filled with microbubble particles and cells, printed and crosslinked on a glass substrate. Fig.17B shows the entire structure of Fig. 17A being encased in a collagen cast gel which provides additional mass and acoustic coupling to enable the ultrasound pulse to penetrate into the filaments and activate the microbubbles rather than just wrapping around the filaments and pushing the filaments through acoustic radiation force.

[0029] Figures 18A-18C. Schematic representation of the bioprinting process and application of focused ultrasound to activate microbubbles within desired regions of the 3D-bioprinted structure. Fig. 18A shows the 3D-bioprinted structure printed using bioinks that incorporate cells and microbubble gene delivery vehicles. Fig. 18B shows the ultrasound focal zone can be passed through the 3D bioprinted structure in user-defined patterns. Fig. 18C shows the plasmid DNA being delivered to cells only in this ultrasound-exposed region causing localized changes in the genetic expression of these cells.

[0030] Figures 19A-19K. Ultrasound-mediated gene delivery in 3D-bioprinted constructs embedded within cast collagen hydrogels. All of this was made possible by embedding the filaments in a cast collagen gel that, while not visible in these images, is supporting the filaments and allows the ultrasound to penetrate into the filaments to activate the microbubbles rather than simply pushing the whole structure around through the acoustic radiation force. Fig. 19A shows a 3D-bioprinted structure comprising two crossing filaments that appear darker than the surrounding collagen gel due to the incorporated microbubbles scattering the light. This helps define the edges of the filaments. Fig. 19B shows ultrasound exposure in the circle region that causes microbubble inertial cavitation and delivery of the DNA payload. The regions of microbubble activation appear clear since the microbubbles are gone. Fig.19C depicts fluorescent images showing the location of cell nuclei and the cells that were transfected withGFP plasmid emit light. Fig. 19D depicts a zoomed-out view showing a whole bioprint and the localized transfection from focused ultrasound exposure. Fig. 19E shows an image of another 3D bioprinted structure in collagen before ultrasound exposure. This one contains multicellular MCF10A breast epithelial spheroid structures that are more complex than the single cells shown in Figs. 19A-19D. Fig.19F shows an image of the gel after ultrasound activation in the circle area. Fig. 19G shows multiple cells were transfected with GFP within a single MCF10A spheroid. Fig.19H shows a magnified image of a GFP-expressing cell from an MCF10A spheroid in the ultrasound focal zone. Fig. 19I shows an additional 3D-bioprinted gel with microbubbles and spheroids containing plasmid DNA for the HER2 oncogene. Fig. 19J shows the gel of Fig. 19I after ultrasound exposure. Fig.19K shows a HER2-expressing cell seen in the spheroid in the ultrasound focal zone. The ability to transfect single cells within the spheroid with HER2 replicates conditions where the very first cells begin to show overexpression of cancer proteins within a healthy tissue.

[0031] Figure 20. Illustration of the collagen embedded filament technique to transfect 3D- bioprinted cells within more complex multicellular spheroid structures. Here, instead of single cells being incorporated into the 3D bioprint, larger and more complex multicellular spheroid structures have been incorporated. These spheroids are surrounded by the DNA-coated microbubbles. Upon ultrasound exposure, only certain cells are activated within the spheroid which replicates the oncogenic expression patterns that initial cancer cells can undergo during the early stages of cancer development. Delivery of the oncogene HER2 is shown in the fluorescence image which localizes to the cell surface. All of this was enabled by embedding the 3D-bioprinted filaments in a cast collagen gel and shows the versatility of applying the technique to different model systems.

[0032] Figures 21A-21D. FRESH bioprinting of cell or spheroid-laden ultrasound-responsive bioinks and subsequent user-defined gene delivery. Fig. 21A shows a schematic depicting bioink composed of ultrasound-responsive microbubbles, single cells or spheroids, and sodium alginate. Fig. 21B shows FRESH bioprinting of ultrasound-responsive bifurcated construct in gelatin-bead support bath slurry containing crosslinking calcium ions. Fig. 21C shows a schematic depicting bioprinting the ultrasound-responsive cell-laden bioink into the support bath followed by release from the support bath and encasing in neutralized collagen with exposure to ultrasound resulting in a localized region of transformed cells. Fig. 21D shows bioprinted filament containing microbubbles and spheroids with user-defined ultrasound exposure resulting in a small subset of transgene-expressing cells.

[0033] Figures 22A-22E. FRESH bioprinting of ultrasound-responsive bioinks. Fig. 22A shows a schematic depicting FRESH bioprinting of a lattice structure into a support bath, and subsequent support bath removal. Fig. 22B shows representative macroscale and brightfield microscopy images of bioprinted alginate grid constructs at 4% w / v with and without the presence of ultrasound-responsive microbubbles incorporated at 2.34x109μB / mL. White triangles indicate filament boundaries. Fig. 22C shows rheological characterization of viscosity versus shear rate comparing alginate bioinks from 2-6% w / v with and without microbubbles (N=3). Figs. 22D and 22E show representative microscopy images of ultrasound-responsive microbubbles coupled to DNA in brightfield (Fig. 22D) and fluorescence (YOYO-1 stain for visualizing plasmid DNA) (Fig. 22E).

[0034] Figures 23A-23E. Ultrasound-mediated microbubble activation and localized transfection in FRESH-printed bioinks. Fig. 23A shows a schematic depicting removal of support bath from bioprinted construct followed by collagen casting and focused ultrasound exposure. Fig. 23B shows representative brightfield microscopy images of bioprinted 4% w / v alginate filaments, increasing in magnification from left to right, before and after application of focused ultrasound. Fig. 23C shows quantification of microbubble activation region in bioprinted alginate filaments upon focused ultrasound application in prints ranging from 2% to 6% w / v alginate (*p<0.05, N=3, One-way ANOVA with Tukey’s post hoc, error bars denote standard deviation). Fig. 23D shows a schematic depicting bioprinting of an alginate lattice construct containing cells and microbubbles before, during, and after focused ultrasound application, resulting in a pattern of secondary gene delivery. Fig. 23E shows bioprinted 4% w / v alginate lattice containing cells and microbubbles coupled with a GFP plasmid, visualized immediately post-printing, after ultrasound application, and 48hr post-printing, with GFP transfected cells and Hoechst nuclear stain.

[0035] Figures 24A-24I. Ultrasound-patterned transfection in multi-ink prints and FRESH- printed tube architectures. Fig. 24A shows a schematic depicting FRESH bioprinting process of multiple bioinks containing microbubbles with different genetic payloads, including support bath removal followed by collagen casting and ultrasound application. Fig. 24B shows multi-ink bioprinted construct showing ultrasound-controlled genetic manipulation in distinct regions with a cross-sectional view. Fig. 24C shows a schematic depicting FRESH bioprinting process of volumetric hollow bifurcated ultrasound-responsive construct, including support bath removal, collagen casting, and ultrasound application. Fig. 24D shows fluorescence microscopy image of bioprinted hollow tubular ultrasound-responsive construct 48hr post-ultrasound, with cells in the ultrasound focal zone overexpressing GFP and Hoechst for nuclear visualization (top and cross-sectional views). Fig. 24E shows brightfield microscopy images of hollow bifurcated ultrasound- responsive construct containing cells and plasmid coupled microbubbles before and after focused ultrasound application. Figs. 24F-24H show macro-scale views of the printed microbubble-containing bifurcated structure prior to collagen incorporation and ultrasound activation. Fig. 24I shows fluorescence microscopy image of the same bioprinted bifurcated architecture shown in Fig. 24E with cells in the ultrasound focal zone overexpressing GFP and Hoechst for nuclear visualization.

[0036] Figures 25A-25D. Ultrasound-mediated localized transfection in FRESH-printed constructs with breast epithelial spheroids. Fig. 25A shows a schematic depicting the printing process of ultrasound-responsive bioinks containing breast epithelial spheroids (MCF10A) including support bath removal followed by collagen casting and ultrasound application resulting in localized transfection of cells in spheroids. Fig. 25B shows MCF10A spheroid-laden 4% w / v RGD-alginate bioprinted filaments containing GFP-coupled microbubbles before ultrasound, 0hr post-ultrasound, and 48hr post-ultrasound with varying ultrasound exposure (10, 40, or 80 pulses). Fig. 25C shows a graph depicting number of transfected cells in a bioprinted spheroid at 48 hr post-ultrasound against varying ultrasound pulses (**p<0.01, ****p<0.0001, N=3, One- way ANOVA with Tukey’s post hoc, error bars denote standard deviation). Fig. 25D shows a schematic depicting a bioprinted filament containing spheroids and HER2 plasmid-coupled microbubbles with localized ultrasound-mediated transfection of cells in spheroids to overexpress HER2. Bioprinted spheroids in the ultrasound focal zone overexpress HER2 with nuclei visualized with Hoechst, with higher magnification images of cells showing characteristic surface HER2 expression (center) and a protrusive morphology (right).

[0037] Figures 26A-26B. Ultrasound-mediated gene delivery in FRESH bioprinted constructs with smaller-diameter filaments. Fig. 26A shows a schematic depicting sodium alginate bioink being loaded with DNA-coupled ultrasound-responsive microbubbles and cells. The bioink is printed into a crosslinking support bath, then incubated to release the bath. Smaller diameter bioprinted filaments are cast in collagen that crosslinks, then the bioprinted construct is exposed to focused ultrasound. After 48 hours, GFP expressing cells are observed only in the region of ultrasound exposure. Fig. 26B shows representative microscopy images of ultrasound- responsive smaller-diameter bioprinted filaments comprising cells and microbubbles coupled with a plasmid for GFP expression before, immediately after, and 48 hours after ultrasound exposure. GFP expressing cells are observed in the region of ultrasound exposure with nuclei labeled (Hoechst).DETAILED DESCRIPTION

[0038] To address the challenge of achieving spatiotemporal control over gene delivery, the present disclosure shows an ultrasound-responsive platform that enables user-defined control over DNA delivery in combination with bioprinted architectures. This allows for an established print to be genetically manipulated remotely and on-demand. Genetic manipulation in tissue constructs has been used to elucidate the function and impact of specific genes. It has been shown that gene functionality and cellular gene expression depends on both the spatiotemporal and architectural context of the matrix.4,16,28,46,68As such, the ability to remotely control genetic manipulation for precise spatial and temporal control within tissue constructs is important for functional characterization of genetic impact on cellular processes and phenotypes.

[0039] Focused ultrasound has several desirable properties as an activating stimulus for triggering gene delivery in tissue including its multi-centimeter tissue penetration depth, biocompatibility and ease of remote application.5,18,23A low-intensity focused ultrasound beam can be used in combination with ultrasound-responsive gene delivery particles, such as gas- core microbubbles23,54, injected intravenously to facilitate gene delivery within a localized tissue region of interest.10,37,43,53,64These microbubbles contain a gas core, typically a mixture of air and low solubility perfluorocarbon gas, stabilized by a lipid monolayer.8,12,42,47Plasmid DNA can be electrostatically coupled to the surface of microbubbles that contain cationic lipids in the lipid monolayer coating.10,43,53,54When focused ultrasound is applied, the microbubbles rapidly oscillate, cavitate, and release their DNA payload.9,22,63The microbubble and ultrasound interaction leads to an effect known as sonoporation9,34,57,67, where nearby cell membranes are transiently disrupted to allow nucleic acid payload to enter the cell, leading to genetic manipulation of the cells in the focal zone. The ultrasound can be spatially focused to a region on the order of a cubic millimeter achieving spatiotemporal control over the ultrasound trigger and minimizing off-target DNA delivery.9,15

[0040] Focused ultrasound has several advantageous characteristics compared to other external stimuli, such as light. Ultrasound has a multi-centimeter penetration depth through cell- dense tissue constructs5in contrast to light which is in the range of multi-millimeter scale penetration.2,69Near infrared (NIR) light, while capable of deeper penetration, has difficulty being focused through a highly scattering medium like a dense tissue construct, while ultrasound has much less scattering and can achieve tight focal zones.

[0041] Here a new coaxial bioprinting platform technology is disclosed that leverages these ultrasound / microbubble interactions to address the current limitations of spatiotemporally controlling genetic manipulation in cell-seeded coaxial bioprinted constructs. A coaxialbioprinting technique, using bioinks containing ultrasound-responsive gene delivery particles, is developed that addresses the unique challenges of multiple fluids being extruded and mixing at the end of the nozzle to achieve microbubble distributions and stability that allows for spatial and temporal control of genetic manipulation (Fig. 1). Echogenic microbubble gene delivery particles and cells are incorporated into a sodium alginate bioink precursor solution in the core of the coaxial needle, with calcium ion crosslinker in the sheath compartment. These solutions are simultaneously printed using the coaxial nozzle so that the alginate bioink is crosslinked upon extrusion to form alginate filaments containing cells and microbubbles. These filaments can be deposited with spatial control to form larger structures. Focused ultrasound can then be applied to a user-defined region of the coaxially bioprinted construct where the echogenic microbubbles oscillate and rupture inducing sonoporation and localized DNA delivery. This facilitates spatiotemporally controllable genetic transfection of cells only in the targeted region.

[0042] The present disclosure demonstrates a newly developed method of coaxially bioprinting ultrasound-responsive microbubbles within cell-laden bioinks to enable spatiotemporal- controlled genetic manipulation in the constructs. The present disclosure also shows the ability to bioprint high-viability cell-laden scaffolds and use focused ultrasound to modulate the size of the DNA delivery zone and number of genetically manipulated cells in a 3D construct. This new coaxial bioprinting platform enables remote-controlled genetic manipulation in printed tissue constructs and can be leveraged for future studies of genetic drivers of disease as well as to remotely guide tissue healing and regeneration through controlled gene expression.

[0043] The use of microbubbles as gene vectors is based on the hypothesis that destruction of DNA-loaded microbubbles by a focused ultrasound beam during their microvascular transit through the target area will result in localized transduction upon disruption of the microbubble shell, while sparing non-targeted areas. In some embodiments, the microbubbles are gene or molecular therapy vectors.

[0044] Additionally, advances in support bath-mediated bioprinting techniques enable freeform fabrication of spatially-complex tissue constructs that mimic tissue architectural features using soft bioinks. However, there remains a need to direct cell processes post-fabrication to model disease or instruct cell behavior through controlled genetic manipulation. Disclosed herein is a new support-bath mediated bioprinting technique, which is developed to fabricate ultrasound- responsive tissue constructs that enable patterning of gene delivery within specified regions of bioprinted architectures using a focused ultrasound beam. This technique leverages interactions of focused ultrasound and responsive microbubble particles embedded in alginate hydrogel bioinks to facilitate ultrasound-modulated transfection of cells and spheroids. Freeform support-bath mediated bioprinting enables formation of hollow tubular architectures that can be genetically manipulated by ultrasound with user-defined spatial control. Exemplary embodiments show that multiple bioink formulations can be activated in a single print, demonstrating spatial patterning of multiple genetic cargoes. Furthermore, functionalizing embedded microbubbles with DNA coding for human epidermal growth factor receptor 2 (HER2) overexpression enables the focused ultrasound to initiate HER2 expression in bioprinted mammary epithelial spheroid cells for cancer modeling applications. Overall, the disclosed platform presents a new tool for ultrasound-programmable gene delivery within intricate constructs printed with soft bioinks with broad utility for directing coordinated cell processes in disease modeling and tissue regeneration.

[0045] Aspects of the current disclosure are now described with additional details and options as follows: (I) Definitions; (II) Compositions and Methods of Use; (III) 3D bioprinting method; (IV) Kits; (V) Examples; and (VI) References. These sections do not limit the interpretation of the disclosure and are provided for organizational purposes only. (I) Definitions

[0046] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated.

[0047] Tissue: As used herein, “tissue” means an aggregate of cells.

[0048] Bioink: As used herein, “bioink” means a liquid, semi-solid, or solid composition for use in bioprinting. In some embodiments, bioink comprises cell solutions, cell aggregates, cell- comprising gels, multicellular bodies, or tissues. In some embodiments, the bioink additionally comprises non-cellular materials that provide specific biomechanical properties that enable bioprinting. In some embodiments the bioink comprises an extrusion compound.

[0049] Bioprinting: As used herein, “bioprinting” means utilizing three-dimensional, precise deposition of cells (e.g., cell solutions, cell-containing gels, cell suspensions, cell concentrations, multicellular aggregates, multicellular bodies, etc.) via methodology that is compatible with an automated or semi-automated, computer-aided, three-dimensional prototyping device (e.g., a bioprinter).

[0050] Scaffold: As used herein, “scaffold” refers to synthetic scaffolds such as polymer scaffolds and porous hydrogels, non-synthetic scaffolds such as pre-formed extracellular matrixlayers, dead cell layers, and decellularized tissues, and any other type of pre-formed scaffold that is integral to the physical structure of the engineered tissue and not able to be removed from the tissue without damage / destruction of said tissue.

[0051] Microparticle or microbubble: The term “microparticle” or “microbubble (μB)” refers to any spherical arrangement of lipids creating an outer shell and an inner void space. The term is used herein to describe microbubbles as encapsulated gas-filled spheres with a diameter in the range from 0.05 to 30 microns, typically with a mean diameter between 1 to 6 μm. The lipid layer of the microbubble may be modified to bind molecules in a stable manner.

[0052] Cavitation: The term “cavitation” is used to describe the process where microbubbles expand and compress upon exposure to ultrasound in the acoustic field. Ultrasound waves propagate through high- and low-pressure cycles, and the pressure differences make the MBs expand during the low-pressure phase and compress during the high-pressure phase. This oscillation can be stable for several cycles (stable cavitation), but it can also end in violent collapse of the MBs (inertial cavitation), depending on the pressure amplitude and frequency.

[0053] Sonoporation: The term “sonoporation”, is used herein to describe the use of sound (typically ultrasonic frequencies) for modifying the permeability of the cell plasma membrane. Sonoporation employs the acoustic cavitation of microbubbles, thus enhancing the delivery of microbubble-coupled cargo to cells and / or at the release site. As used herein, the term “cargo” is understood to include the delivery of a desired nucleic acid molecule or plasmid DNA to a bioprinted cell construct.

[0054] Transfection: The term “transfection” or “transfected” refers to the introduction of foreign DNA into a cell to form a transgenic cell.

[0055] Gene: As used herein, the term “gene” means the deoxyribonucleotide sequences comprising the coding region of a structural gene and including sequences located adjacent to the coding region on both the 5′ and 3′ ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The term “gene” encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.”

[0056] Hydrogel: “Hydrogel” refers to a class of polymeric materials which are swollen in an aqueous medium, but which do not dissolve in water. Hydrogels are highly absorbent (they can contain over 99% water) natural or synthetic polymers. Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content. A hydrogel is defined as a substance formed when an organic polymer (natural or synthetic) is cross-linkedvia covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure which entraps water molecules to form a gel. Examples of materials which can be used to form a hydrogel include polysaccharides such as alginate, polyphosphazines, and polyacrylates, which are crosslinked ionically, or block copolymers such as Pluronics™ or Tetronics™, polyethylene oxide (PEO)-polypropylene glycol (PPG) block copolymers which are crosslinked by temperature or pH, respectively. In embodiments herein, alginate can be ionically cross-linked with divalent cations, in water, at room temperature, to form a hydrogel matrix. In some embodiments, the hydrogel is a natural hydrogel material, such as agarose, methylcellulose, hyaluronan, and other naturally derived polymers.

[0057] Crosslinking: “Crosslinking” as generally used herein means the formation of more than one covalent linkage within or between molecules. For example, gels are substantially dilute crosslinked systems, which exhibit no flow when in the steady state. By weight, gels are mostly liquid, yet they behave like solids due to a three-dimensional crosslinked network within the liquid. It is the crosslinks within the fluid that give a gel its structure (hardness) and contribute to stickiness (tack). In some examples, for hydrogel crosslinking, this can also be achieved by non-covalent linkages, including ionic interactions, hydrogen bonding, and physical entanglements.

[0058] Close proximity: The term “close proximity” is used herein to describe a distance between microparticles / microbubbles and target cells / mammalian cells within the filament structures of the hydrogel scaffold or bioprinted construct. In embodiments of the present disclosure, this distance ranges from about 0 μm to about 35 μm. In some embodiments, the microbubbles may be pre-coupled to the target cells / mammalian cells through electrostatic interaction prior to printing the filaments such that the distance between the cells and the microbubbles is effectively 0 μm.

[0059] Focused region or focal zone: The term “focused region” or “focal zone” is used herein to describe a region within the hydrogel scaffold, where an application of a focused ultrasound activates the microbubbles causing cavitation which delivers nucleic acids to nearby target cells through sonoporation. The lower limit of the focal zone size may be dependent on the ultrasound frequency. In embodiments, a range of dimensions for focal zone cross-section is from about 0.3 mm to about 2 mm. In some embodiments, a larger area of effect may be created by rastering the focal zone through the desired volume, such that any size / portion of a tissue construct can be exposed. Bioprinting

[0060] In some embodiments, at least one component of the engineered tissues or cell constructs is bioprinted. In further embodiments, bioprinted constructs are made with a method that utilizes a rapid prototyping technology based on three-dimensional, automated, computer- aided deposition of cells, including cell solutions, cell suspensions, cell-comprising gels or pastes, cell concentrations, multicellular bodies (e.g., cylinders, spheroids, ribbons, etc.), and, optionally, confinement material onto a biocompatible support surface (e.g., composed of hydrogel and / or a porous membrane) by a three-dimensional delivery device (e.g., a bioprinter). In some embodiments, the term “engineered,” when used to refer to tissues or constructs means that cells, cell solutions, cell suspensions, cell-comprising gels or pastes, cell concentrates, multicellular aggregates, and layers thereof are positioned to form three- dimensional structures by a computer-aided device (e.g., a bioprinter) according to a computer script. In further embodiments, the computer script is, for example, one or more computer programs, computer applications, or computer modules including executable instructions. In still further embodiments, three-dimensional tissue structures form through the post-printing fusion of cells or multicellular bodies which, in some cases, is similar to self-assembly phenomena in early morphogenesis.

[0061] While a number of methods are available to arrange cells, cell aggregates, and cell- containing materials on a biocompatible surface to produce a three-dimensional structure, including manual placement, positioning by an automated, computer-aided machine such as a bioprinter is advantageous. Advantages of delivery of cells, cell aggregates, and cell-containing materials with this technology include rapid, accurate, and reproducible placement of cells or multicellular bodies to produce constructs exhibiting planned or pre-determined orientations or patterns of cells, cell aggregates and / or layers thereof with various compositions. Advantages also include assured high cell density, while minimizing cell damage.

[0062] In some embodiments, the method of bioprinting is continuous and / or substantially continuous. A non-limiting example of a continuous bioprinting method is to dispense bio-ink (i.e., cells, cells combined with an extrusion compound, or aggregates of cells) from a bioprinter via a dispense tip (e.g., a syringe, needle, capillary tube, etc.) connected to a reservoir of bio- ink. In further non-limiting embodiments, a continuous bioprinting method is to dispense bio-ink in a repeating pattern of functional units. In various embodiments, a repeating functional unit has any suitable geometry, including, for example, circles, squares, rectangles, triangles, polygons, and irregular geometries, thereby resulting in one or more tissue layers with planar geometry achieved via spatial patterning of distinct bio-inks and / or void spaces. In further embodiments, a repeating pattern of bioprinted function units comprises a layer and a pluralityof layers are bioprinted adjacently (e.g., stacked) to form an engineered tissue with laminar geometry. In various embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more layers are bioprinted adjacently (e.g., stacked) to form an engineered tissue or a three-dimensional cell construct. In further embodiments, one or more layers of a tissue with laminar geometry also has planar geometry.

[0063] In some embodiments, continuous bioprinting facilitates printing larger tissues or cell constructs from a large reservoir of bio-ink, optionally using a syringe mechanism. Continuous bioprinting is also a convenient way to co-print spatially-defined boundaries, using an extrusion compound, a hydrogel, a polymer, bio-ink, or any printable material that is capable of retaining its shape post-printing; wherein the boundaries that are created are optionally filled in via the bioprinting of one or more bio-inks, thereby creating a mosaic tissue with spatially-defined planar geometry.

[0064] In some embodiments, methods in continuous bioprinting involve optimizing and / or balancing parameters such as print height, pump speed, robot speed, or combinations thereof independently or relative to each other. In certain cases, the bioprinter head speed for deposition is 3 mm / s, with a dispense height of 0.5 mm for the first layer and dispense height may be increased 0.4 mm for each subsequent layer. In some embodiments, the dispense height is approximately equal to the diameter of the bioprinter dispense tip. Without limitation, a suitable and / or optimal dispense distance does not result in material flattening or adhering to the dispensing needle. In various embodiments, the bioprinter dispense tip has an inner diameter of about, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 μm, or more, including increments therein. In various embodiments, the bio-ink reservoir of the bioprinter has a volume of about 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 cubic centimeters, or more, including increments therein. The pump speed, in some cases, is suitable and / or optimal when the residual pressure build-up in the system is low. Favorable pump speeds, in some cases, depend on the ratio between the cross-sectional areas of the reservoir and dispense needle with larger ratios requiring lower pump speeds. In some embodiments, a suitable and / or optimal print speed enables the deposition of a uniform line without affecting the mechanical integrity of the material.

[0065] In some embodiments, the speed and scalability of the techniques and methods disclosed herein are utilized to design, build, and operate commercial facilities for production of 3D cell constructs and / or disease models for use in generation of cell-based tools for research and development, such as in vitro assays. In further embodiments, the 3D cell constructs and / ordisease models are produced, stored, distributed, marketed, advertised, and sold as, for example, cellular arrays (e.g., microarrays or chips), tissue arrays (e.g., microarrays or chips), and kits for biological assays and high-throughput drug screening. In other embodiments, the 3D cell constructs and / or disease models are produced and utilized to conduct biological assays (e.g., transfection assays) and / or drug screening as a service. Bioink

[0066] Disclosed herein, in certain embodiments, are three-dimensional, living tissues or cell constructs, including HEK293T cells, breast epithelial spheroid, osteoblast cells, and methods that comprise bioprinting cells. In some embodiments, cells are bioprinted by depositing or extruding bioink from a bioprinter. In some embodiments, “bioink” includes liquid, semi-solid, or solid compositions comprising a plurality of cells. In some embodiments, bioink comprises liquid or semi-solid cell solutions, cell suspensions, or cell concentrations. In further embodiments, a cell solution, suspension, or concentration comprises a liquid or semi-solid (e.g., viscous) carrier and a plurality of cells. In still further embodiments, the carrier is a suitable cell nutrient media, such as those described herein. In some embodiments, bioink comprises a plurality of cells that optionally cohere into multicellular aggregates prior to bioprinting. In further embodiments, bio- ink comprises a plurality of cells and is bioprinted to produce a specific planar and / or laminar geometry.

[0067] In some embodiments, the bioink is produced by collecting a plurality of cells in a fixed volume; wherein the cellular component(s) represent at least about 30% and at most about 100% of the total volume. In some embodiments, bioink comprises semi-solid or solid multicellular aggregates or multicellular bodies. In further embodiments, the bioink is produced by 1) mixing a plurality of cells or cell aggregates and a biocompatible liquid or gel in a pre- determined ratio to result in bioink, and 2) compacting the bioink to produce the bioink with a desired cell density and viscosity. In some embodiments, the compacting of the bioink is achieved by centrifugation, tangential flow filtration (“TFF”), or a combination thereof.

[0068] In some embodiments, the bioinks disclosed herein are characterized by high cellularity by volume, e.g., a high concentration of living cells. In further embodiments, the bioinks comprise at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400 or more million cells per milliliter of solution. In a particular embodiment, the bioinks comprise about 50 to about 300 million cells / mL. In some embodiments, bioinks that have high cellularity by volume are used to bioprint engineered tissues and constructs with highcell density. In further embodiments, the engineered tissues and constructs are at least about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more percent cells.

[0069] In some embodiments, the compacting of the bioink results in a composition that is extrudable, optionally allowing formation of multicellular aggregates or multicellular bodies. In some embodiments, “extrudable” means able to be shaped by forcing (e.g., under pressure) through a nozzle or orifice (e.g., one or more holes or tubes). In some embodiments, the compacting of the bioink results from growing the cells to a suitable density.

[0070] In some embodiments, the cells of the bioink are not cohered and / or adhered. In other embodiments, the cells of the bioink are cohered and / or adhered. In some embodiments, “cohere,” “cohered,” and “cohesion” refer to cell-cell adhesion properties that bind cells, multicellular aggregates, multicellular bodies, and / or layers thereof. In further embodiments, the terms are used interchangeably with “fuse,” “fused,” and “fusion.” In some embodiments, the bioink additionally comprises support material, cell culture medium (or supplements thereof), extracellular matrix (or components thereof), cell adhesion agents, cell death inhibitors, anti- apoptotic agents, anti-oxidants, extrusion compounds, and combinations thereof.

[0071] In some embodiments, the bioink comprises cancer cells (e.g., tumor cells). In further embodiments, the cancer cells are cells of one or more cell lines. In some embodiments, the bioink comprises HEK293T cells, breast epithelial spheroid, or osteoblast cells. Extrusion compounds

[0072] In some embodiments, the bioink comprises an extrusion compound (i.e., a compound that modifies the extrusion properties of the bioink). Examples of extrusion compounds include, but are not limited to gels, hydrogels, peptide hydrogels, amino acid-based gels, surfactant polyols (e.g., Pluronic F-127 or PF-127), thermo-responsive polymers, hyaluronates, alginates, extracellular matrix components (and derivatives thereof), collagens, gelatin, other biocompatible natural or synthetic polymers, nanofibers, and self-assembling nanofibers.

[0073] Suitable hydrogels include those derived from collagen, GelMA (gelatin methacryloyl), methacrylated collagen, hyaluronate, hyaluronan, fibrin, alginate, agarose, chitosan, gellan gum, cellulose, hyaluronic acid, and combinations thereof. In some embodiments, suitable hydrogels include PEG-based hydrogels, decellularized extracellular matrix hydrogels, and Matrigel. In other embodiments, suitable hydrogels are synthetic polymers. In further embodiments, suitable hydrogels include those derived from poly(acrylic acid) and derivatives thereof, poly(ethylene oxide) and copolymers thereof, poly(vinyl alcohol), polyphosphazene, and combinations thereof. In various specific embodiments, the confinement material is selected from: hydrogel, NovoGel®, agarose, alginate, gelatin, Matrigel™, hyaluronan, poloxamer, peptide hydrogel,poly(isopropyl n-polyacrylamide), polyethylene glycol diacrylate (PEG-DA), hydroxyethyl methacrylate, polydimethylsiloxane, polyacrylamide, poly(lactic acid), silicon, silk, or combinations thereof. In some embodiments, pluronic may be used as a sacrificial bioink.

[0074] In some embodiments, hydrogel-based extrusion compounds are crosslinkable gels. In further embodiments, crosslinkable gels include those crosslinkable by chemical means. For example, in some embodiments, suitable hydrogels include alginate-containing crosslinkable hydrogels. In various embodiments, suitable hydrogels comprise about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more percent alginate. In some embodiments, following bioprinting, constructs are optionally incubated with an agent to chemically crosslink the hydrogel, such as a solution of CaCl2,in order preserve a bioprinted architecture prior to cohesion of the cells. Further, in some embodiments, the bioprinted constructs are optionally incubated with alginate lyase to enzymatically degrade the hydrogel. In further embodiments, the bioprinted constructs are optionally incubated with alginate lyase at a concentration of about 0.2-0.5 mg / ml to enzymatically degrade the hydrogel.

[0075] In some embodiments, suitable hydrogels include gelatin. In various embodiments, suitable hydrogels comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more percent gelatin.

[0076] In some embodiments, the concentration of gelatin is about 5-15% and the concentration of alginate is about 0.5-5% in the extrusion compound or hydrogel. In a particular embodiment, the concentration of gelatin is 10% and the concentration of alginate is 1% in the extrusion compound or hydrogel.

[0077] In some embodiments, hydrogel-based extrusion compounds are thermoreversible gels (also known as thermo-responsive gels or thermogels). In some embodiments, a suitable thermoreversible hydrogel is not a liquid at room temperature. In specific embodiments, the gelation temperature (Tgel) of a suitable hydrogel is about 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, including increments therein. In certain embodiments, the Tgel of a suitable hydrogel is about 10°C to about 40°C. In further embodiments, the Tgel of a suitable hydrogel is about 20°C to about 30°C. In some embodiments, the bioink (e.g., comprising hydrogel, one or more cell types, and other additives, etc.) described herein is not a liquid at room temperature. In some embodiments, a suitable thermoreversible hydrogel is not a liquid at mammalian body temperature. In specific embodiments, the gelation temperature (Tgel) of a suitable hydrogel is about 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C,40°C, 41°C, 41°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, including increments therein. In certain embodiments, the Tgel of a suitable hydrogel is about 22°C to about 52°C. In further embodiments, the Tgel of a suitable hydrogel is about 32°C to about 42°C. In some embodiments, the bioink (e.g., comprising hydrogel, one or more cell types, and other additives, etc.) described herein is not a liquid at mammalian body temperature. In specific embodiments, the gelation temperature (Tgel) of a bioink described herein is about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, including increments therein.

[0078] Polymers composed of polyoxypropylene and polyoxyethylene form thermoreversible gels when incorporated into aqueous solutions. These polymers have the ability to change from the liquid state to the gel state at temperatures maintainable in a bioprinter apparatus. The liquid state-to-gel state phase transition is dependent on the polymer concentration and the ingredients in the solution.

[0079] In some embodiments, the viscosity of the hydrogels and bioinks presented herein is measured by any means described. For example, in some embodiments, an LVDV-II+CP Cone Plate Viscometer and a Cone Spindle CPE-40 are used to calculate the viscosity of the hydrogels and bio-inks. In other embodiments, a Brookfield (spindle and cup) viscometer is used to calculate the viscosity of the hydrogels and bioinks. In some embodiments, the viscosity ranges referred to herein are measured at room temperature. In other embodiments, the viscosity ranges referred to herein are measured at body temperature (e.g., at the average body temperature of a healthy human).

[0080] In further embodiments, the hydrogels and / or bioinks are characterized by having a viscosity of between about 500 and 1,000,000 centipoise, between about 750 and 1,000,000 centipoise; between about 1000 and 1,000,000 centipoise; between about 1000 and 400,000 centipoise; between about 2000 and 100,000 centipoise; between about 3000 and 50,000 centipoise; between about 4000 and 25,000 centipoise; between about 5000 and 20,000 centipoise; or between about 6000 and 15,000 centipoise.

[0081] In some embodiments, the non-cellular components of the bioink (e.g., extrusion compounds, etc.) are removed prior to use. In further embodiments, the non-cellular components are, for example, hydrogels, peptide hydrogels, amino acid-based gels, surfactant polyols, thermo-responsive polymers, hyaluronates, alginates, collagens, or other biocompatible natural or synthetic polymers. In still further embodiments, the non-cellular components are removed by physical, chemical, or enzymatic means. In some embodiments, a proportion of the non-cellular components remain associated with the cellular components at the time of use.Microbubbles

[0082] The microbubbles contain a gas core surrounded by an envelope formed from one or more lipids and one or more emulsifying agents in the form of a lipid monolayer or multilayer. The outer surface of the envelope forms a protective film.

[0083] The envelope is in the form of a lipid film, in the form of a monolayer or multilayer, preferably in the form of a monolayer. The lipid film may be between 1 and 100 nm thick, preferably between 1 and 10 nm thick, most preferably between 2 and 5 nm thick. In one embodiment, the lipid film is a monolayer that is about 10 nm thick. The overall charge for the lipid envelope may be neutral, positive or negative. The envelope may contain one or more emulsifying agents. The emulsifying agent generally contains a hydrophilic portion, typically a hydrophilic polymer, and a hydrophobic portion. The emulsifying agent or a portion thereof, generally the hydrophilic portion of the emulsifying agent, forms a protective border on the outer surface of the microbubble. The border may be in the form of a brush where the hydrophilic portion of the emulsifying agent extends from the lipid containing portion of the envelope to form a border on the outer surface of the microbubble.

[0084] A variety of lipids may be used to form the lipid film. The lipids may be natural or synthetic. Suitable lipids include phospholipids, fatty acids, triacyl glycerols, sphingolipids, terpenes, and waxes. Preferably the envelope contains one or more phospholipids. The lipid envelope may contain lipids with acyl chains of varying lengths and degrees of saturation. The lipid envelope may contain lipids with a single acyl chain length, or different lipids with different acyl chain lengths. In some embodiments, the lipid is a long-chain lipid, preferably a saturated diacyl phosphatidylcholine (Di-Cn—PC, where n is between 12 and 24, preferably where n is 16 or 18). Suitable lipids include phosphocholines, phosphoglycerols, phosphatidic acids, phosphoethanolamines, and phosphoserines. Examples include 1,2-Dilauroyl-sn-Glycero-3- Phosphocholine (dilauroylphosphatidylcholine, DLPC), 1,2-Dimyristoyl-sn-Glycero-3- Phosphocholine (dimyristoylphosphatidylcholine, DMPC), 1,2-Dipentadecanoyl-sn-Glycero-3- Phosphocholine (dipentadecanoylphosphatidylcholine, DPDPC), 1,2-dipalmitoyl-sn-Glycero-3- Phosphocholine (dipalmitoylphosphatidylcholine, DPPC), 1-Myristoyl-2-Palmitoyl-sn-Glycero-3- Phosphocholine (1-myristoyl-2-palmitoylphosphatidylcholine, MPPC), 1,2-Dimyristoyl-sn- Glycero-3-[Phospho-rac-(1-glycerol)] (DMPG), 1,2-Dimyristoyl-3-Trimethylammonium-Propane, cholesterol and its derivatives, fatty acids, fatty alcohols, and fatty esters.

[0085] Lipids in the envelope may have different acyl chain lengths. The number of carbons in the acyl chains of the lipids may range from 10 to 24 carbons. The average acyl chain length of the lipids in microbubbles typically ranges from 10 to 24 carbons. For example, the average acylchain length of the lipids may be 20, 18, 16, 14, 13, or 12 carbons. The envelope may include one or more synthetic lipids with asymmetric acyl chains, where one acyl chain is longer than another. Lipids with longer acyl chain lengths are generally preferred compared to lipids with shorter chain lengths. Generally, lipids with longer chain lengths produce more microbubbles with a greater shelf life. However, the chain length should not be too long. Generally, longer chained lipids (e.g. 24-carbon vs.16-carbon) are more resistant to oxygen passage. Resistance to gas release is also increased in walls composed of saturated (i.e. no double bonds) versus unsaturated lipids. Lipids with one or more double bonds contain kinks in the acyl chains due to the presence of the double bonds, which creates irregularities in the packing geometry, and thereby allows for gas to transfer out of the microbubble more rapidly. A lipid film comprising lipids having longer chain lengths may have decreased permeability to gases compared to one with lipids with shorter acyl chain lengths. This decreased permeability may be attributed to an increase in attractive dispersion and hydrophobic forces between the hydrophobic tails of adjacent lipid molecules, resulting in a more cohesive lipid film. However, longer acyl chains generally provide greater envelope cohesion, which can improve mechanical strength and reduced gas escape kinetics. In some applications for the microbubbles may contain short acyl chain lengths (e.g. ≤C14). Shorter acyl chain lengths are generally more unstable than longer acyl chains.

[0086] Emulsifying agent(s) refers to the one or more surfactants in the envelope that contain a molecule aiding lipid adsorption to the gas / liquid interface and stabilizing the microbubble to prevent coalescence. Typically, the surfactant is a hydrophilic polymer attached to a hydrophobic anchor via one or more covalent bonds. Preferably the hydrophobic anchor is a lipid. The hydrophobic anchor may be an alkyl group, in the form of a single chain or multiple chains. Typically, the alkyl group is 12 to 24 carbons in length. Alternatively, hydrophobic anchors such as sterols, or polymers such as polycaprolactone may be used. The hydrophilic polymer in the emulsifying agent is polyethylene glycol (PEG). Typical weight average molecular weights for PEG range from about 550 Da to 5,000 Da. Alternatively, other molecules can be in place of PEG. Alternatives include polypropylene glycol, polyvinyl alcohol, poly-N-vinyl pyrrolidone and copolymers thereof, mixed polyalkylene oxides having a solubility of at least one gram / liter in aqueous solutions such as some poloxamer nonionic surfactants, neutral water-soluble polysaccharides, including dextran, Ficoll, and derivatized celluloses, non-cationic poly(meth)acrylates, non-cationic polyacrylates, such as poly(meth)acrylic acid, and esters amide and hydroxyalkyl amides thereof, and combinations thereof.

[0087] The envelope may contain a variety of different amounts of base lipids and emulsifyingagents. An optimum ratio of emulsifying agents to base lipids, which lies between a minimum ratio needed to have sufficient amounts of emulsifying agents to aid in lipid adsorption, shield the surface of the microbubble and prevent coalescence and a maximum ratio where lateral repulsion forces due to the presence of the emulsifying agent begin to significantly disrupt packing of the base lipid, may be determined experimentally.

[0088] Optionally, the envelope may include one or more molecules in addition to the lipid(s) and emulsifying agent(s) to stabilize the microbubbles. Suitable stabilizers include polymers and proteins. Suitable polymers include lipophilic and amphiphilic polymers. The proteins may be a single protein or a mixture of proteins. Suitable proteins include lipophilic and amphiphilic proteins. Examples of protein envelopes include, but are not limited to, albumin. The proteins and polymers may be within the lipid monolayer or multilayer. Examples of multilayer envelopes include, but are not limited to, polyelectrolyte multilayers.

[0089] The gas core contains at least one gas or gas precursor. The gas must be pharmacologically acceptable, i.e. biocompatible and have minimal toxicity when released. The gas precursor is a material that forms a gas in the microbubbles. Suitable gas precursors include volatile liquids. The gas may be a fluorinated gas, such as fluorinated gases include CF4, C2F4, C2F6, C3F6, C3F8, C4F8, C4F10, and SF6. The gas may be a perfluorocarbon gas, such as CF4, C2F4, C2F6, C3F6, C3F8, C4F8, and C4F10. n-Perfluorobutane (C4F10) is an insoluble gas that will not condense at the temperature of use and is pharmacologically acceptable. Other suitable gases include carbon dioxide, nitrogen, nitrous oxide, helium, argon, nitric oxide, xenon, carbon monoxide, oxygen, and volatile anesthetics, such as isoflurane. These gases may be in the gas core alone or in combination with one or more other gases. The amount of gas contained within the microparticles will depend on the type of gas.

[0090] The microbubbles can be of any suitable size for carrying or transporting the substance. In some embodiments, the microbubbles can have a size (e.g., diameter) of between about 0.2 μm-5 μm. In some embodiments, the microbubbles can have a size of about 1 μm-5 μm.

[0091] In some embodiments, the microbubbles can be made of a substance to which the material that is to be delivered can attach or be bound. As noted in embodiments herein, the microbubbles can have an electrostatic charge that can facilitate electrostatic attachment of a substance with an opposite charge (e.g., negatively charged DNA binding to a positively charged lipid, or a negatively charged envelope binding to a positively charged substance). In some embodiments, the substance that is to be delivered can be covalently bound to the microbubbles via any suitable covalent bond or chemical group. Also, preferably the microbubbles are formed such that upon application of ultrasound, the bubbles will cavitate,burst, collapse, etc. in order that the substance for delivery can enter into a cell.

[0092] The methods, compositions, systems, kits and apparatuses can be used to deliver a wide variety of substances to a cell of interest using ultrasound. In general, the substance that is to be delivered can be loaded into, onto or attached or at least partially bound to a microbubble. As noted above, that attachment can be accomplished via any suitable mechanism such as for example, an electrostatic attraction or an ionic bond, a covalent bond, hydrogen bonding, van der Waals forces, conjugation, and the like. Non-bound substance can be washed away or otherwise removed or can be left in the solution if desired.

[0093] In some embodiments, cells can be transformed by delivery of nucleic acid molecules. Examples of nucleic acid molecules include without limitation DNA, RNA, linear DNA, circular DNA, cDNA, miRNA, shRNA, siRNA, as well as constructs of any of the same such as vectors, plasmids, viral constructs (e.g., CMV, AAV, CaMV, SV40, lentivirus, retrovirus), and the like. The nucleic acid molecule can be or include a plasmid, a transposase construct, a viral nucleic acid construct, a nucleic acid encoding a protein, a polypeptide, amino acid, or a peptide.

[0094] In some embodiments, peptides, polypeptide, amino acid or a protein can be delivered. Examples of such substances may include, without limitation, an antibody, an antibody fragment, an enzyme, a cytokine, a chemokine, an antigen, a neurotrophin, a hormone, a fluorescent protein, an interleukin, a growth factor, a toxin, a signal sequence, a transcription factor, a reporter molecule, a promoter, a polypeptide or protein whose coding sequence is optimized for cellular expression. The nucleic acid, peptide, polypeptide or protein can be loaded into or onto the microbubble and then delivered e.g., by destroying the microbubble in proximity to the target cell such that the substance can contact and enter the target cell.

[0095] Other substances can be delivered as well. Non-limiting examples include lipids, fatty acids, therapeutic compounds (e.g., small molecules, biologics), and active compounds or molecules.

[0096] The delivery substance can be present in any suitable amount or concentration. For example, the substance (e.g., nucleic acid molecule, protein, peptide, or polypeptide) may be present at a concentration ranging from about 1 to 10,000 μg / mL of culture or microbubble solution volume, or any range, sub range, or value there between. In some embodiments, the substance (e.g., nucleic acid molecule) can be coated onto the microbubbles at a concentration of 1 μg to 4 mg per 1 mL final volume (e.g., microbubble volume) or any value or sub range there between. In some aspects, the substance (e.g., nucleic acid) can be present at a concentration of 1 mg nucleic acid per 1 mL final volume or any value or sub range there between. In some embodiments herein, nucleic acid molecule such as plasmid DNA may bepresent at a concentration of 0.1 pg per microbubble.

[0097] In some embodiments, in place of or in addition to nucleic acid molecule / polypeptide, one or more drugs (i.e., pharmaceutically active agents) may be delivered using the disclosed compositions and methods. Suitable classes of active agents include, but are not limited to, antibiotic agents, antimicrobial agents, anti-acne agents, antibacterial agents, antifungal agents, antiviral agents, steroidal anti-inflammatory agents, non-steroidal anti-inflammatory agents, anesthetic agents, antipruriginous agents, antiprotozoal agents, anti-oxidants, antihistamines, and vitamins. The one or more drugs may be encapsulated within the microbubbles or loaded onto the microbubbles for delivery to the target. Ultrasound Targeted Microbubble Cavitation

[0098] The microbubbles can be cavitated using ultrasound as described herein. One example for illustration purposes is ultrasound targeted microbubble cavitation. It is a technique for transformation of a cell or tissue in which a substance such as bioactive molecules, e.g., negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubbles. In vitro, these vector-carrying microbubbles can simply be added to the media / hydrogel matrix containing the cells of interest. The DNA delivery to the target cell occurs by acoustic cavitation at a resonant frequency of the microbubbles. The mechanical energy generated by the microbubble destruction results in transient pore formation in the target cells. As a result of this sonoporation effect, the transfection efficiency into and across the target cells is enhanced.

[0099] Any suitable ultrasound instrument may be used with the methods, systems, kits, and compositions described herein. Some embodiments relate to the use of a custom ultrasound instrument capable of complete control of all ultrasonic parameters needed to achieve thorough optimization. For example, such an instrument can be configured to detect the efficiency of the cavitation in real-time with use of an attached hydrophone. The device further can be configured to modulate frequency, and mechanical index (MI), for example. In terms of varying the ultrasound frequency, a range of 0.5 MHz to 15 MHz can be utilized or controlled, or any sub range or value there between. A frequency within such a range can effectively resonate the microbubbles. The length of each ultrasound pulse can range from 100 μs to 100 ms. A total of 10-80 ultrasound pulses may be utilized to effectively activate microbubbles for gene delivery. Mechanical index is essentially an overall estimate of power that a given set of ultrasound parameters will transmit. A higher MI is likely to produce a larger bio-effect. In some embodiments, MI can be varied from a benign measure of 0.1 to a maximal energy of 5.0, orany sub range or value there between. [000100] Some embodiments may utilize a continuous waveform of focused or unfocused ultrasound. For example, continuous wave blasts through the microbubbles results in delivery of nucleic acid / peptide molecules to the target cells. An ultrasonic transducer may be brought into sufficient proximity with the hydrogel matrix containing microbubbles and target cells for the microbubbles / target cells to be contacted with the ultrasonic waves to effectuate transfection. In some embodiments, an arbitrary waveform of ultrasound can be used as well. For example, a wave pulse can be generated and can allow for on / off cycling (i.e., duty cycle). As such the target cells can move directly into the path of the waveform generated. In some embodiments, some or all of the cavitation might occur at the place where the target cells meet the waveform. [000101] Ultrasound targeted microbubble cavitation can induce a number of biophysical effects, including; cavitation (microbubble collapse), radiant pressure (force created by the ultrasound waves), and microstreaming (sheer forces created by oscillating microbubbles). The primary effect is mediated by cavitation. Cavitation can be further defined as the growth, oscillation and collapse of microbubbles with the application of an acoustic field. Microbubble cavitation results in mechanical induction of transient pores that can be 100 nm in size and a few seconds half-life. Cavitation can also be divided into stable and inertial categories. Stable cavitation occurs when the microbubbles oscillate stably in a low intensity acoustic field, generating sheer force and microstreaming. Inertial cavitation occurs within a high intensity acoustic field when the microbubbles rapidly expand and then collapse, producing a shock wave and microenvironments of extreme pressure and temperature. Varying the pulse duration or the length of time that the cavitation frequency is applied results in a relatively higher amount of cavitation, increasing overall efficiency of transfection per experiment. [000102] Inertial cavitation is the process wherein a void or microbubble in a matrix rapidly collapses, producing a shock wave. Microscopic gas bubbles that are generally present in a hydrogel matrix will be forced to oscillate due to an applied acoustic field. If the acoustic intensity is sufficiently high, the bubbles will first grow in size and then rapidly collapse. The effect of different transducer frequencies on ultrasound targeted microbubble cavitation efficiency using different frequencies (0.5, 1, 2.25, 3, 5, 10, and 15 MHz) can be assessed. These are common frequencies used in microbubble cavitation experiments in this disclosure. The microbubble preparations used in some of the experiments described herein range from 1-5 μm size. In some aspects, the higher frequencies can work, but in some cases may be less efficient at generating the inertial cavitation for some of the microbubble sizes specifically listed herein for some embodiments. Additionally, different-sized microbubble populations may usedifferent frequency ranges to achieve effective inertial cavitation. The frequencies can be generated by any suitable ultrasound apparatus, including for example, one using focused or unfocused transducers. Mechanical index may be used as an overall estimate of sonographic power that a given set of ultrasound parameters will convey. [000103] The cell / tissue that is transfected or has had a substance delivered to it can undergo further downstream processing or analysis. For example, the methods can further include culturing, sub culturing, visualizing, imaging, utilizing as a seed culture, propagated, further treated or transformed, purified, and / or have one or more products purified or removed from it. (II) Compositions and Methods of Use [000104] In the present disclosure, the development of coaxially-3D bioprinted ultrasound- responsive scaffolds for remote-controlled gene delivery is described. This configuration uniquely enables ultrasound-mediated genetic manipulation of cells via microbubble gene delivery vehicles embedded within coaxially-3D bioprinted cell-laden filaments. This ultrasound- mediated gene delivery technique has an advantage in 3D tissue scaffolds over traditional gene delivery methods that use vectors, such as viruses or lipofectamine, because the diffusion of these vectors is inhibited by the dense scaffold matrix making it a challenge to evenly deliver the DNA to cells in a controlled and predictable way. Diffusion mechanisms are also a challenge to localize to a desired region making it difficult for the user to target a particular region of cells to transfect. With diffusion, it is also difficult to control the timing of when the traditional vectors will reach a desired region of the gel. The ultrasound triggered microbubble delivery technique addresses these challenges by incorporating the DNA loaded microbubbles in the liquid bioink achieving an even distribution across the whole bioprinted construct. The focused ultrasound trigger gives the user spatiotemporal control over when and where the cells are transfected. [000105] In one embodiment, the present invention contemplates a microbubble population that can be useful for delivering all types of oligonucleotides, including but not limited to, deoxyribonucleic acid (DNA) vectors, antisense, RNAi, miRNA, and / or transcription factor decoys. [000106] The present disclosure also provides a range of alginate concentrations, microbubble concentrations, and number of focused ultrasound pulses that are amenable to ultrasound- mediated genetic manipulation in the bioprinted constructs. Additionally, there is an optimal range of microbubble concentration and number of focused ultrasound pulses to control the number of manipulated cells and the diameter of the zone containing the transfected cells. The present disclosure demonstrates a bioprinting method with the ability to modulateaforementioned parameters to uniquely control the number of genetically manipulated cells, spatial aspects of the region containing these transformed cells, and the timepoint where genetic manipulation occurs. This contrasts with standard methods of gene delivery in thick 3D constructs, which lack both spatial and temporal control due to challenges of vector diffusion. [000107] The compositions and methods of the present disclosure enables controlled genetic manipulation of cells in a variety of advanced biofabrication applications. The versatility of coaxial bioprinting allows the simultaneous and controlled deposition of multiple bioinks in the form of filaments, as described here, and also allows hollow tube structures to be created by switching the sacrificial crosslinker bioink to the core of the coaxial needle. Additionally, concentric multi-material deposition enables the co-printing of bioinks that have desirable complimentary mechanical and biological properties, and the co-printing and controlled deposition of multiple cell types in a single construct.40Printing constructs that allow controlled genetic manipulation of incorporated epithelial, stromal, and immune cells19,59would enable researchers to recapitulate important aspects of cellular interactions within the tissue microenvironment. [000108] The compositions and methods of the present disclosure enable users to define when and where different genes can be overexpressed and can be used to study the role that genes of interest play in a variety of different biological processes including the maturation of tissue, wound healing, and disease progression. Targeted gene delivery could be used to spatially- control stimulated differentiation of stem cells as well as manipulate patient-derived cells to investigate disease progression and test drugs for patient-specific treatment options. A noteworthy potential application of this technology is the ability to manipulate a subset of established cells in a bioprinted microenvironment to overexpress oncogenes for modeling cancer progression in the 3D tissue context.11,45,55As sonoporation is a physical gene delivery method, as opposed to chemical or biological methods, this approach will likely be amenable to many cell types, including cells that are considered hard-to-transfect by chemical methods, as well as primary cells, stem cells, and non-immortalized cell lines. Additional optimization of ultrasound parameters such as the intensity or number of pulses applied may be required for these additional cell types. Thus, this technology may enable the application of controlled genetic perturbation techniques to investigate cell-crosstalk, migration, differentiation, and disease progression among other possibilities. [000109] Disclosed herein is a composition comprising a hydrogel scaffold with a plurality of filament structures printed on a substrate, and a collagen cast gel that encases the plurality of filament structures of the hydrogel scaffold, wherein the composition is configured to receive apopulation of microparticle-coupled cargo for controlled delivery to one or more target cells by application of ultrasound energy. In some embodiments, the filament structures are printed adjacent to each other and / or on top of one another such that the filament structures form a cross-bridge. In some embodiments, the filament structures are printed in any suitable geometry selected from the group of circles, squares, rectangles, triangles, polygons, laminar geometries, and irregular geometries, or any combinations thereof. [000110] In embodiments, the method or order of crosslinking the filaments may affect the degree of interaction between filaments that are crossing / touching. If the filaments are fully crosslinked as they are printed, there would be less interaction between the filaments. However, if the filaments are crosslinked together, then more physical interaction may occur. In some embodiments, the cells inside the filaments may interact with cells in neighboring filaments through secretion of signaling molecules and may even migrate in between two crosslinked filaments. The addition of the collagen casting layer holds the filaments in place and keeps them physically touching each other. [000111] In disclosed embodiments, the hydrogel scaffold comprises an agent selected from the group of alginate, fibrin, agarose, chitosan, gellan gum, cellulose, hyaluronic acid, gelatin methacryloyl, and methacrylated collagen, or any combinations thereof. In disclosed embodiments, the collagen cast gel is bioprinted. In embodiments, at least one of the filament structures from the plurality of filament structures of the hydrogel scaffold further comprises the population of microparticle-coupled cargo. In embodiments, the population of microparticle- coupled cargo is distributed over an area of at least one of the filament structures of the hydrogel scaffold. In some embodiments, the population of microparticle-coupled cargo comprises a population of gas-filled microbubbles coupled to a substance selected from the group of nucleic acid, peptide, polypeptide, amino acid, protein, and drug, or any combinations thereof. In embodiments disclosed herein, the substance is coupled to the gas-filled microbubbles via an electrostatic interaction, covalent bond, hydrogen bond, van der Waals force, or conjugation. In embodiments, the one or more target cells comprise mammalian single cells or cell aggregates dispersed in at least one of the filament structures of the plurality of filament structures of the hydrogel scaffold. In some embodiments, the population of microparticle-coupled cargo and the one or more target cells present in the hydrogel scaffold are in close proximity to each other. In embodiments, the application of ultrasound energy causes cavitation of the microparticles at a focused region of the hydrogel scaffold to deliver the cargo to the one or more target cells in proximity to the cavitated microparticles within thehydrogel scaffold. A schematic of the ultrasound induced sonoporation and localized delivery of DNA to cells within a 3D hydrogel construct is shown in Fig. 16. [000112] In some embodiments, it is possible to have some filaments with cells and microbubbles and others without any combination of those. It can be used to help localize the effects of transfection from the ultrasound to just the printed regions of interest. This can also be used to achieve transfection in regions below the diffraction limit of the ultrasound that determines the focal zone size. In some examples, the compositions may even include different microbubbles in different adjacent filaments showing ultrasound activation of two alternating DNA transfections, for example, where alternating filaments had microbubbles carrying different DNA payloads. [000113] Disclosed herein is a composition comprising a hydrogel scaffold with a plurality of filament structures, a collagen cast gel encasing the plurality of filament structures of the hydrogel scaffold, a population of mammalian cells incorporated into the hydrogel scaffold, and a plurality of microbubbles dispersed throughout the filament structures of the hydrogel scaffold, wherein the microbubbles are attached to one or more nucleic acid molecules to be expressed in the mammalian cells. A schematic of final bioprinting construct embedded in cast collagen gel is shown in Figs. 17A and 17B. In some embodiments, the hydrogel scaffold comprises an agent selected from the group of alginate, fibrin, agarose, chitosan, gellan gum, cellulose, hyaluronic acid, gelatin methacryloyl, and methacrylated collagen, or any combinations thereof. In some cases, the hydrogel scaffold comprises sodium alginate. In embodiments, the collagen cast gel is selected from the group of type I collagen, type II collagen, and type III collagen, or any combinations thereof. In some cases, the collagen cast gel comprises type I collagen. In embodiments, the plurality of microbubbles and the population of mammalian cells incorporated into the hydrogel scaffold are in close proximity to each other. In some embodiments, the plurality of microbubbles dispersed throughout the filament structures of the hydrogel scaffold is electrostatically attached to a surface of the mammalian cells incorporated into the hydrogel scaffold. [000114] In embodiments, each microbubble of the plurality of microbubbles comprises an envelope and a gas core. In some cases, the envelope of each microbubble comprises a lipid monolayer or multilayer. In embodiments, the gas core of each microbubble comprises a gas selected from the group of perfluorocarbon, carbon dioxide, nitrogen, nitrous oxide, helium, argon, nitric oxide, xenon, carbon monoxide, oxygen, and isoflurane, or any combinations thereof. In some cases, the gas core of each microbubble comprises perfluorocarbon gas. In embodiments, each microbubble of the plurality of microbubbles has a diameter ranging fromabout 0.05 μm to about 5 μm. Exemplary embodiments may include a microbubble diameter ranging from about 0.05-0.2 μm, 0.05-0.3 μm, 0.05-0.5 μm, 0.1-0.3 μm, 0.1-0.5 μm, 0.2-0.5 μm, 0.3-0.5 μm, 0.4-0.6 μm, 0.5-0.8 μm, 0.5-1 μm, 0.7-1 μm, 0.05-1 μm, 1-2 μm, 1-3 μm, 1-4 μm, 1- 5 μm, 2-4 μm, 2-5 μm, 3-4 μm, 3-5 μm, or 4-5 μm. Thus, the diameter of the microbubble may be 0.05 μm, 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, or 5 μm. In a preferred embodiment, each microbubble of the plurality of microbubbles has a diameter ranging from about 1 μm to about 5 μm. [000115] In embodiments, the one or more nucleic acid molecules attached to the microbubbles are selected from the group of a linear DNA, circular DNA, cDNA, miRNA, shRNA, siRNA, plasmid, viral construct, and transposase construct, or any combinations thereof. In some cases, the one or more nucleic acid molecules are attached to the microbubbles via electrostatic interaction. In embodiments, the population of mammalian cells comprises single cells scattered throughout the filament structures of the hydrogel scaffold. In some cases, the single cells of the population of mammalian cells comprise HEK293T cells. In some cases, the single cells of the population of mammalian cells comprise osteoblasts. In embodiments, the population of mammalian cells comprises cell aggregates incorporated throughout the filament structures of the hydrogel scaffold. In some cases, the cell aggregates comprise spheroids or organoids. In some cases, the spheroids comprise breast epithelial spheroids. [000116] In some embodiments, the composition is configured to be responsive to an ultrasonic frequency ranging from about 0.5 MHz to about 15 MHz. Exemplary embodiments may include an ultrasonic frequency ranging from about 0.5-2 MHz, 0.5-3 MHz, 0.5-5 MHz, 1-3 MHz, 1-5 MHz, 2-5 MHz, 3-6 MHz, 5-8 MHz, 5-10 MHz, 7-10 MHz, 8-12 MHz, 10-15 MHz, or 12-15 MHz. Thus, the ultrasonic frequency may be 0.5 MHz, 1 MHz, 1.5 MHz, 2 MHz, 2.5 MHz, 3 MHz, 3.5 MHz, 4 MHz, 4.5 MHz, 5 MHz, 5.5 MHz, 6 MHz, 6.5 MHz, 7 MHz, 7.5 MHz, 8 MHz, 8.5 MHz, 9 MHz, 9.5 MHz, 10 MHz, 10.5 MHz, 11 MHz, 11.5 MHz, 12 MHz, 12.5 MHz, 13 MHz, 13.5 MHz, 14 MHz, 14.5 MHz, or 15 MHz. In embodiments, the ultrasonic frequency causes the microbubbles to implode thereby delivering the one or more nucleic acid molecules to the mammalian cells for expression. In some cases, the delivery of the one or more nucleic acid molecules to the mammalian cells occurs at a localized region of the hydrogel scaffold. [000117] Disclosed herein is a composition comprising a hydrogel scaffold with a plurality of filament structures, a collagen cast gel encasing the plurality of filament structures of the hydrogel scaffold, a population of mammalian cells incorporated into the hydrogel scaffold, a plurality of microbubbles dispersed throughout the filament structures of the hydrogel scaffold,and a plurality of nucleic acid molecules distributed over an area of at least one of the filament structures of the hydrogel scaffold to be expressed in the mammalian cells. In some embodiments, the mammalian cells, the plurality of microbubbles and the plurality of nucleic acid molecules are in close proximity to each other within the filament structures of the hydrogel scaffold. In some embodiments, the mammalian cells, the microbubbles and the nucleic acid molecules present within the filament structures of the hydrogel scaffold are devoid of any interactions. In some embodiments, the microbubbles are attached to the nucleic acid molecules via an electrostatic interaction to form a microbubble-coupled cargo. In some cases, the microbubble-coupled cargo lacks interaction with the mammalian cells within the hydrogel scaffold. In some examples, the microbubble-coupled cargo is further attached to a surface of the mammalian cells within the hydrogel scaffold via electrostatic interaction. In some cases, a distance between the mammalian cells and the microbubbles within the hydrogel scaffold ranges from about 0 μm to about 35 μm. Exemplary embodiments may include a distance ranging from about 0-3 μm, 0-5 μm, 0-10 μm, 2-5 μm, 3-6 μm, 5-8 μm, 5-10 μm, 5-15 μm, 9-12 μm, 10-15 μm, 10-20 μm, 12-15 μm, 15-18 μm, 15-20 μm, 15-25 μm, 20-23 μm, 20-25 μm, 20- 30 μm, 22-25 μm, 25-28 μm, 25-30 μm, 25-35 μm, 30-33 μm, 30-35 μm, or 32-35 μm. Thus, the distance between the mammalian cells and the microbubbles within the hydrogel scaffold may be 0 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm, 30 μm, 31 μm, 32 μm, 33 μm, 34 μm, or 35 μm. [000118] In embodiments, the population of mammalian cells comprises single cells or cell aggregates incorporated into the filament structures of the hydrogel scaffold. In embodiments, the composition is configured to be responsive to an ultrasound frequency ranging from about 0.5 MHz to about 15 MHz. Exemplary embodiments may include an ultrasound frequency ranging from about 0.5-2 MHz, 0.5-3 MHz, 0.5-5 MHz, 1-3 MHz, 1-5 MHz, 2-5 MHz, 3-6 MHz, 5- 8 MHz, 5-10 MHz, 7-10 MHz, 8-12 MHz, 10-15 MHz, or 12-15 MHz. Thus, the ultrasound frequency may be 0.5 MHz, 1 MHz, 1.5 MHz, 2 MHz, 2.5 MHz, 3 MHz, 3.5 MHz, 4 MHz, 4.5 MHz, 5 MHz, 5.5 MHz, 6 MHz, 6.5 MHz, 7 MHz, 7.5 MHz, 8 MHz, 8.5 MHz, 9 MHz, 9.5 MHz, 10 MHz, 10.5 MHz, 11 MHz, 11.5 MHz, 12 MHz, 12.5 MHz, 13 MHz, 13.5 MHz, 14 MHz, 14.5 MHz, or 15 MHz. In some embodiments, the ultrasound frequency causes cavitation of the microbubbles at a focal zone to deliver the nucleic acid molecules to the mammalian cells in proximity to the cavitated microbubbles for expression within the hydrogel scaffold. In embodiments, a size of the focal zone of the hydrogel scaffold ranges from about 0.3 mm to about 2 mm. Exemplary embodiments may include a size of the focal zone ranging from about0.3-0.6 mm, 0.3-0.8 mm, 0.3-1 mm, 0.4-0.8 mm, 0.5-0.8 mm, 0.5-1 mm, 0.5-1.5 mm, 1-1.3 mm, 1-1.5 mm, 1-1.8 mm, 1-2 mm, 1.3-1.6 mm, 1.3-1.8 mm, 1.4-1.8 mm, 1.5-1.8 mm, or 1.5-2 mm. Thus, the size of the focal zone of the hydrogel scaffold may be 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, or 2 mm. [000119] Also disclosed herein is a method of creating a transgenic target cell, the method comprising the steps of: (a) providing a hydrogel scaffold with a plurality of filament structures encased in a collagen cast gel, wherein the filament structures comprise a plurality of target cells and a population of ultrasound-responsive microbubbles stably bound to one or more nucleic acid molecules; (b) applying ultrasound to a focused region of the hydrogel scaffold with sufficient energy to cavitate the microbubbles causing an uptake of the one or more nucleic acid molecules by at least one target cell from the plurality of target cells present in proximity to the cavitated microbubbles within the focused region of the hydrogel scaffold; and (c) imaging the ultrasound-exposed hydrogel scaffold to visualize the at least one target cell that comprises the one or more nucleic acid molecules; wherein the at least one target cell expressing the one or more nucleic acid molecules is the transgenic target cell. A schematic representation of the bioprinting process and application of focused ultrasound to activate microbubbles within desired regions of the 3D-bioprinted structure is shown in Figs.18A-18C. Ultrasound-mediated gene delivery of a GFP plasmid in 3D-bioprinted constructs is shown in Figs. 19A-19D. [000120] In some examples, the hydrogel scaffold is bioprinted coaxially. In embodiments, the hydrogel scaffold comprises a bioink selected from the group of alginate, fibrin, agarose, chitosan, gellan gum, cellulose, hyaluronic acid, gelatin methacryloyl, and methacrylated collagen, or any combinations thereof. In some cases, the hydrogel scaffold comprises alginate bioink. In embodiments, the alginate bioink is printed with a printing pressure ranging from about 10 kPa to about 15 kPa. Exemplary embodiments may include a printing pressure ranging from about 10-12 kPa, 10-13 kPa, 10-14 kPa, 11-13 kPa, 11-14 kPa, 11-15 kPa, 12-14 kPa, 12-15 kPa, or 13-15 kPa. Thus, the printing pressure may be 10 kPa, 11 kPa, 12 kPa, 13 kPa, 14 kPa, or 15 kPa. [000121] In some examples, each microbubble of the population of ultrasound-responsive microbubbles comprises an envelope and a gas core. In embodiments, the envelope of each microbubble comprises a lipid monolayer or multilayer. In embodiments, the gas core of each microbubble comprises a perfluorocarbon gas. In some cases, the perfluorocarbon gas further comprises a gas selected from the group of CF4, C2F4, C2F6, C3F6, C3F8, C4F8, and C4F10, or any combinations thereof. In embodiments, each microbubble has a diameter ranging from about0.05 μm to about 5 μm. Exemplary embodiments may include a microbubble diameter ranging from about 0.05-0.2 μm, 0.05-0.3 μm, 0.05-0.5 μm, 0.1-0.3 μm, 0.1-0.5 μm, 0.2-0.5 μm, 0.3-0.5 μm, 0.4-0.6 μm, 0.5-0.8 μm, 0.5-1 μm, 0.7-1 μm, 0.05-1 μm, 1-2 μm, 1-3 μm, 1-4 μm, 1-5 μm, 2- 4 μm, 2-5 μm, 3-4 μm, 3-5 μm, or 4-5 μm. Thus, the diameter of the microbubble may be 0.05 μm, 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, or 5 μm. In a preferred embodiment, each microbubble of the plurality of microbubbles has a diameter ranging from about 1 μm to about 5 μm. [000122] In embodiments, the one or more nucleic acid molecules bound to the microbubbles are selected from the group of a linear DNA, circular DNA, cDNA, miRNA, shRNA, siRNA, plasmid, viral construct, and transposase construct, or any combinations thereof. In some examples, smaller plasmids show better diffusion into the nearby target cell during the sonoporation process. In embodiments disclosed herein, plasmids that are about 5,563 base pairs long can be readily delivered through sonoporation. However, plasmids that are beyond 10 times larger than the disclosed plasmid size may have trouble fitting through the small holes created in the cell membrane during sonoporation. This may be addressed by using a supercoiled DNA to make it smaller. Most desirable genes can fit on plasmids that are 50,000 base pairs long or shorter. In examples disclosed herein, the nucleic acids (such as siRNA, microRNAs, and so on) are coupled to the cationic microbubbles used for this method. In some cases, the one or more nucleic acid molecules are coupled to the microbubbles via electrostatic interaction. [000123] In embodiments, a distance between the target cells and the microbubbles within the filament structures of the hydrogel scaffold ranges from about 0 μm to about 35 μm. Exemplary embodiments may include a distance ranging from about 0-3 μm, 0-5 μm, 0-10 μm, 2-5 μm, 3-6 μm, 5-8 μm, 5-10 μm, 5-15 μm, 9-12 μm, 10-15 μm, 10-20 μm, 12-15 μm, 15-18 μm, 15-20 μm, 15-25 μm, 20-23 μm, 20-25 μm, 20-30 μm, 22-25 μm, 25-28 μm, 25-30 μm, 25-35 μm, 30-33 μm, 30-35 μm, or 32-35 μm. Thus, the distance between the target cells and the microbubbles within the hydrogel scaffold may be 0 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm, 30 μm, 31 μm, 32 μm, 33 μm, 34 μm, or 35 μm. [000124] In some embodiments, applying ultrasound comprises applying an ultrasonic frequency ranging from about 0.5 MHz to about 15 MHz. Exemplary embodiments may include an ultrasonic frequency ranging from about 0.5-2 MHz, 0.5-3 MHz, 0.5-5 MHz, 1-3 MHz, 1-5MHz, 2-5 MHz, 3-6 MHz, 5-8 MHz, 5-10 MHz, 7-10 MHz, 8-12 MHz, 10-15 MHz, or 12-15 MHz. Thus, the ultrasonic frequency may be 0.5 MHz, 1 MHz, 1.5 MHz, 2 MHz, 2.5 MHz, 3 MHz, 3.5 MHz, 4 MHz, 4.5 MHz, 5 MHz, 5.5 MHz, 6 MHz, 6.5 MHz, 7 MHz, 7.5 MHz, 8 MHz, 8.5 MHz, 9 MHz, 9.5 MHz, 10 MHz, 10.5 MHz, 11 MHz, 11.5 MHz, 12 MHz, 12.5 MHz, 13 MHz, 13.5 MHz, 14 MHz, 14.5 MHz, or 15 MHz. In some cases, applying ultrasound further comprises an application of a plurality of ultrasound pulses, wherein the plurality of ultrasound pulses ranges from about 10 pulses to about 80 pulses. Exemplary embodiments may include ultrasound pulses ranging from about 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 20-30, 20-40, 20-50, 20- 60, 20-70, 20-80, 30-40, 30-50, 30-60, 30-70, 30-80, 40-50, 40-60, 40-70, 40-80, 50-60, 50-70, 50-80, 60-70, 60-80, or 70-80 pulses. Thus, the ultrasound pulses that may be applied includes 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 pulses. In embodiments, a length of each ultrasound pulse of the plurality of ultrasound pulses ranges from about 0.1 ms to about 100 ms. Exemplary embodiments may include a length of ultrasound pulse ranging from about 0.1-5 ms, 0.1-10 ms, 0.1-20 ms, 0.1-50 ms, 5-10 ms, 5-20 ms, 5-30 ms, 5-50 ms, 10-20 ms, 10- 40 ms, 10-50 ms, 20-30 ms, 20-50 ms, 30-40 ms, 30-50 ms, 30-60 ms, 40-50 ms, 40-60 ms, 40- 80 ms, 50-60 ms, 50-80 ms, 50-100 ms, 60-70 ms, 60-80 ms, 60-100 ms, 70-80 ms, 70-100 ms, 80-90 ms, 80-100 ms, or 90-100 ms. Thus, the length of each ultrasound pulse may be 0.1 ms, 0.5 ms, 1 ms, 2 ms, 5 ms, 10 ms, 15 ms, 20 ms, 25 ms, 30 ms, 40 ms, 50 ms, 60 ms, 70 ms, 80 ms, 90 ms, or 100 ms. [000125] In embodiments of the present disclosure, 2.25 MHz of ultrasonic frequency resonates with the 1-5 μm microbubbles used in the studies, which enables their inertial cavitation necessary for the DNA delivery. Inertial cavitation may be achieved with these microbubbles at other frequencies, if high enough intensities are used. A different frequency range may be used for a different sized microbubble population. The ultrasound frequency range disclosed herein is from about 0.5 MHz to about 15 MHz. The length of the pulse can change from about 0.1 ms to about 100 ms. In some examples, the pulse may be kept relatively short to reduce any potential heating of the tissue that might occur. A good duty cycle may include 1 pulse per second that can prevent heating; however, in some examples, it may also be increased up to 3 pulses per second. In yet other examples, it may be reduced to 1 pulse every 5 seconds. In embodiments, the pulses may be kept below 80 total pulses. The ultrasound pulse range disclosed herein is from about 10 pulses to about 80 pulses. In some examples, the lower end of the range could potentially be broadened to 1 pulse; however, this may be less efficient at activating enough microbubbles for gene delivery.[000126] In embodiments, a size of the focused region where ultrasound is applied within the hydrogel scaffold ranges from about 0.3 mm to about 2 mm. Exemplary embodiments may include a size of the focused region ranging from about 0.3-0.6 mm, 0.3-0.8 mm, 0.3-1 mm, 0.4- 0.8 mm, 0.5-0.8 mm, 0.5-1 mm, 0.5-1.5 mm, 1-1.3 mm, 1-1.5 mm, 1-1.8 mm, 1-2 mm, 1.3-1.6 mm, 1.3-1.8 mm, 1.4-1.8 mm, 1.5-1.8 mm, or 1.5-2 mm. Thus, the size of the focused region of the hydrogel scaffold may be 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, or 2 mm. In embodiments, the target cells comprise mammalian single cells or cell aggregates dispersed throughout the filament structures of the hydrogel scaffold. In some embodiments, the uptake of the one or more nucleic acid molecules by at least one target cell within the focused region of the hydrogel scaffold is caused by an inertial cavitation of the microbubbles upon ultrasound application. In some examples, the number of transfected cells may be modulated by varying the number of focused ultrasound pulses applied. In some embodiments, the size region over which DNA is delivered may be modulated by varying the density of microbubbles within the printed filaments. In some embodiments, imaging the ultrasound-exposed hydrogel scaffold comprises visualizing and measuring a diameter of a zone comprising the transgenic target cells along the filament structures. [000127] Also disclosed herein is a composition prepared by a process comprising the steps of: (a) fabricating a bioink for coaxial bioprinting; (b) incorporating a plurality of microbubbles into the fabricated bioink; (c) incorporating a population of mammalian cells into the fabricated bioink comprising the microbubbles; (d) bioprinting the fabricated bioink comprising the microbubbles and the mammalian cells to form a construct with a plurality of filament structures; and (e) bioprinting a collagen cast gel embedding the plurality of filament structures of the construct to form the composition. [000128] In some examples, the bioink comprises alginate. In some examples, the alginate is used at a concentration of from about 2% to about 6% weight by volume. Exemplary embodiments may include a concentration of alginate ranging from about 2-4%, 2-5%, 3-5%, 3- 6%, or 4-6% weight by volume. Thus, alginate may be used at a concentration of 2%, 3%, 4%, 5% or 6% weight by volume. In some cases, the alginate bioink is printed with a printing pressure ranging from about 10 kPa to about 15 kPa. Exemplary embodiments may include a printing pressure ranging from about 10-12 kPa, 10-13 kPa, 10-14 kPa, 11-13 kPa, 11-14 kPa, 11-15 kPa, 12-14 kPa, 12-15 kPa, or 13-15 kPa. Thus, the printing pressure may be 10 kPa, 11 kPa, 12 kPa, 13 kPa, 14 kPa, or 15 kPa.[000129] In some examples, the bioink comprises gelatin. In some cases, the gelatin bioink is printed with a printing pressure ranging from about 40 kPa to about 60 kPa. Exemplary embodiments may include a printing pressure ranging from about 40-43 kPa, 40-45 kPa, 43-48 kPa, 45-50 kPa, 40-50 kPa, 50-53 kPa, 50-55 kPa, 53-58 kPa, 55-60 kPa, or 50-60 kPa. Thus, the printing pressure may be 40 kPa, 41 kPa, 42 kPa, 43 kPa, 44 kPa, 45 kPa, 46 kPa, 47 kPa, 48 kPa, 49 kPa, 50 kPa, 51 kPa, 52 kPa, 53 kPa, 54 kPa, 55 kPa, 56 kPa, 57 kPa, 58 kPa, 59 kPa, or 60 kPa. In embodiments, bioprinting the fabricated bioink further includes an addition of a crosslinking agent (e.g., calcium chloride). [000130] In embodiments, the microbubbles are ultrasound responsive. In some embodiments, the mammalian cells comprise single cells or cell aggregates dispersed throughout the filament structures of the construct. In some examples, the microbubbles are dispersed throughout the filament structures of the construct and are located in close proximity to the mammalian cells within the construct. In embodiments, a distance between the mammalian cells and the microbubbles within the filament structures of the construct ranges from about 0 μm to about 35 μm. Exemplary embodiments may include a distance ranging from about 0-3 μm, 0-5 μm, 0-10 μm, 2-5 μm, 3-6 μm, 5-8 μm, 5-10 μm, 5-15 μm, 9-12 μm, 10-15 μm, 10-20 μm, 12-15 μm, 15- 18 μm, 15-20 μm, 15-25 μm, 20-23 μm, 20-25 μm, 20-30 μm, 22-25 μm, 25-28 μm, 25-30 μm, 25-35 μm, 30-33 μm, 30-35 μm, or 32-35 μm. Thus, the distance between the mammalian cells and the microbubbles within the filament structures of the construct may be 0 μm, 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm, 30 μm, 31 μm, 32 μm, 33 μm, 34 μm, or 35 μm. [000131] In some examples, the microbubbles are coupled to a substance selected from the group of a nucleic acid, peptide, polypeptide, amino acid, protein, and drug, or any combinations thereof. In embodiments, the collagen cast gel is selected from the group of type I collagen, type II collagen, and type III collagen, or any combinations thereof. In embodiments, each filament structure of the plurality of filament structures of the construct has a size ranging from about 150 μm to about 700 μm in diameter. Exemplary embodiments may include a diameter of a cross- section of each filament structure ranging from about 150-200 μm, 150-250 μm, 150-300 μm, 150-400 μm, 150-500 μm, 150-600 μm, 200-300 μm, 200-400 μm, 200-500 μm, 200-600 μm, 200-700 μm, 250-650 μm, 300-400 μm, 300-500 μm, 300-600 μm, 300-700 μm, 350-650 μm, 400-500 μm, 400-600 μm, 400-700 μm, 450-650 μm, 500-600 μm, 500-700 μm, or 600-700 μm. Thus, the diameter of the cross-section of each filament structure may be 150 μm, 180 μm, 200μm, 220 μm, 250 μm, 280 μm, 300 μm, 320 μm, 350 μm, 380 μm, 400 μm, 420 μm, 450 μm, 480 μm, 500 μm, 520 μm, 550 μm, 580 μm, 600 μm, 620 μm, 650 μm, 680 μm, or 700 μm. [000132] Thus, the present disclosure provides a novel coaxial 3D bioprinting technique for ultrasound-controlled gene delivery in cell-laden bioprinted constructs. By incorporating ultrasound-responsive particles into extrudable bioinks and exposing the bioprinted constructs to focused ultrasound, the method successfully demonstrated sonoporation of embedded cells, resulting in targeted DNA delivery and robust ultrasound-controlled transgene expression. Varying the density of ultrasound-responsive microbubble gene delivery vehicles and the number of applied focused ultrasound pulses allows for the modulation of the number of transfected cells and the region of the print where transgene is delivered in the bioprinted constructs. This platform enables remote-controlled genetic manipulation in coaxially bioprinted tissue constructs with the ability to spatiotemporally-define DNA delivery. This technology may enable studies in tissue constructs coaxially printed with multiple bioinks and cell types. The ability to spatiotemporally-control genetic manipulation within coaxially-bioprinted constructs can be leveraged in studies to actuate intercellular communication, guide stem cell differentiation and model disease states, with important applications in understanding disease progression and directing tissue regeneration. [000133] Some of the technical effects and advantages of using the disclosed technology is that it enables spatiotemporally controllable gene delivery in user-defined patterns within 3D- bioprinted structures. This feature allows cells within the construct to interact naturally by self- assembling into different cellular structures as they would in a real tissue, before being perturbed by the genetic manipulation allowing us to study the role that genes play in different aspects of biology and disease. The cast collagen gel holds the 3D-bioprinted filaments in place without causing distortion to the filament structures or to the cellular structures that have formed within the filament. The cast collagen gel enables better coupling of the ultrasound energy into the filament than the surrounding water allowing for more effective microbubble activation. The disclosed compositions and methods are noninvasive and do not disturb the cellular structures within the construct. Different types of microbubbles that respond to different ultrasound pulses could be multiplexed together to allow for delivery of different DNA plasmids at different times in the same and in different locations within the construct. The ultrasound energy allows for genetic manipulation of cells even within a spheroid structure without having to alter the spheroid structure. Traditional / existing methods may require the spheroids to undergo dissociation followed by multiple selection cycles which is undesirable for patient-derived samples (this large amount of manipulation may cause confounding changes to the normalcellular behavior of the spheroid). The use of 3D bioprinting provides advantages over non- bioprinted cast gel systems by being able to build defined architectures consisting of multiple cell types and the ability to control the deposition of multiple materials. The bioprinting method of the present disclosure uniquely preserves the stability of the gas-filled microbubbles while still allowing for the formation of complex 3D architectures in the final bioprinted construct. The disclosed compositions and methods work with several different bioprinting techniques including coaxial and FRESH / support-bath mediated bioprinting. [000134] Biological tissues exhibit both intricate structural complexity as well as dynamic, temporally complex cell behaviors that occur within these environments. Rapidly advancing biofabrication techniques have been able to create ever more structurally complex and biomimetic tissues by enabling spatial control over matrix properties, geometry, and embedded cell type. These three-dimensional cell environments are important because they provide greater physiological relevancy compared to 2D cultures for modeling cellular interactions with other cells and their surrounding matrix, as well as response to biochemical cues. There is a need to match this controlled structural complexity with controlled temporal complexity of cellular behavior to non-invasively direct cell processes throughout these structures after they have been formed and throughout their maturation. [000135] Freeform Reversible Embedding of Suspended Hydrogels (FRESH) 3D bioprinting fabrication method, as disclosed herein, may be used for creating relevant matrix architectures via spatially controlled deposition of desired cell types and chemical cues. FRESH bioprinting utilizes extrusion printing of bioinks into a dissolvable gelatin-bead support bath that maintains construct geometry and allows printing of intricate structures such as hollow vessel networks, bone grafts, and complex cardiac structures. A particular advantage of FRESH bioprinting is the ability to create complex geometries using soft bioinks, such as collagen or alginate, which cannot structurally support themselves due to gravity when uncrosslinked. [000136] The temporal complexity of cell behavior is coordinated within the spatial complexity provided by FRESH bioprinted structures. To model dynamic cellular processes, the ability to manipulate protein presentation of selected cells within bioprinted scaffolds through targeted gene expression is key. For example, in HER2+ breast cancer as described in the exemplary embodiments, only a single cell or small subset of cells initially begins to overexpress HER2 protein, and the interactions between transformed and healthy cells are very important to model. While the creation of 3D tissues using bioprinting is widely characterized, it remains a challenge to genetically manipulate cells within these engineered constructs with spatial and temporal specificity. Current strategies include bioprinting scaffolds that incorporate gene complexes,growth factors, or previously manipulated cells into the bioink itself to create a tissue construct that will have sustained release of the gene or factor initially added to the bioink. However, while the existing strategies allow for spatial control of gene delivery, they do not allow for on-demand temporal control of genetic manipulation. Stimuli-responsive materials can be used to biofabricate tissue constructs, followed by application of an applied stimulus to induce cellular or material changes in specific regions. While a number of stimuli-responsive materials have been explored for bioprintable inks in 4D printing strategies, focused ultrasound remains relatively unexplored as a stimulus for activating targeted genetic changes in bioprinted constructs, despite its compatibility with remote manipulation. [000137] Focused ultrasound has been used as an activating stimulus for inducing gene delivery in vivo, demonstrating multi-centimeter penetration depth and well-characterized biocompatibility. Ultrasound-responsive microparticles, such as gas-core lipid-shell microbubbles as described herein, can be utilized for targeted gene therapy applications. Upon ultrasound exposure, the microbubbles begin to rapidly oscillate, cavitate, and release the coupled genetic cargo. Additionally, the microbubble-ultrasound interaction leads to sonoporation, where cells in the focal region undergo transient disruption of the cell membrane, allowing the genetic payload to enter. [000138] Disclosed herein is a technique to fabricate stimuli-responsive constructs using alginate bioinks and support bath-mediated FRESH bioprinting that enables ultrasound- localized genetic manipulation of cells with user-defined spatial and temporal control. Incorporating ultrasound-responsive microbubbles into the bioinks and activating with ultrasound post-fabrication allows for regional sonoporation resulting in robust ultrasound- mediated transgene expression of different genes in distinct regions of complex tissue structures. This approach overcomes issues of vector diffusion through thick hydrogel layers while maintaining print fidelity for creating complex architectures. Multiple concentrations of alginate were bioprinted with high fidelity and the number of transfected cells were modulated by varying ultrasound pulse application. By bioprinting inks with ultrasound-responsive microbubbles loaded with different DNA plasmids, the ability to pattern expression of different genes were shown in the same printed construct with tight localization and minimal off-target effects in the exemplary embodiments. Further, precise ultrasound-mediated transfection of hollow tubular architectures was demonstrated, illustrating the applicability of this technique to genetically manipulate the complex spatially-heterogeneous tissue architectures printable with freeform support-bath mediated techniques. Extending the newly developed technique to a breast epithelial spheroid model, as shown here, a controlled modulation of the number ofactivated spheroid cells with ultrasound was achieved and locally induced HER2 overexpression in a small subset of cells within the printed spheroids was also achieved. [000139] Thus, this platform enables remote-controlled genetic manipulation of cells and spheroids via DNA delivery in FRESH bioprinted tissue constructs, which the user can spatiotemporally define. Leveraging this technique, further studies can investigate the incorporation and controlled genetic manipulation of multiple bioprinted cell types to stimulate controlled intracellular communication within spatially-defined biofabricated microenvironments, enabling new studies of disease initiation and progression, including cancer. Additionally, this technique can be applied to guide regenerative processes such as stem cell differentiation and directed growth factor delivery for tissue regeneration. Altogether, this ultrasound-programmable gene delivery technology can be a powerful tool to perturb biological systems and controllably manipulate cell processes in the context of spatially-defined tissue microenvironments. [000140] Furthermore, the printing parameters of pressure and speed of various needle gauges for extrusion bioprinting of 4% w / v alginate filaments with and without the presence of ultrasound-responsive microbubbles was assessed in embodiments. Different needle gauges were utilized to modulate feature sizes to enhance the bioprinted structure functionality. Higher viscosity biomaterials may be limited to larger needle diameters and lower bioprinted construct resolution, while smaller diameter needle might require excessive pressures that are detrimental for cellular viability and may have a higher chance of needle clogging. As such, 4% w / v alginate was chosen for its relatively low viscosity and shear thinning properties, with relevance in tissue engineering applications. These characteristics lower the material’s viscosity when exposed to shear forces resulting in less extrusion pressure important to achieve target filament diameter. Thus, 4% w / v alginate bioinks can be bioprinted with a wide range of needle sizes with optimized printing parameters. In embodiments, when refining the printing parameters for alginate bioinks, the lowest pressure combined with the highest speed that produced filament diameters similar to theoretical values were chosen. Lower extrusion pressures can reduce the shear stress cells experience, and the faster print speeds minimize the time the cells are outside their optimal environment such as an incubator. In embodiments, when bioprinting with the smaller diameter needle gauges (25G, 27G, and 30G) the print fidelity and filament width was similar when increasing pressure, so the lowest pressure was chosen. However, the smaller diameter needle gauges were limited to a lower optimal speed of 2mm / s compared to the larger diameter needles (20G, 22G) that could be printed at a speed of 4mm / s. [000141] The bioprinting pressure and speed influence filament diameter when using larger gauge needles (such as 20G) but did not impact diameter in smaller gauge needles (such as30G). This trend continued regardless of the presence of ultrasound-responsive microbubbles in the bioink. As such, for a variety of bioprinting needle gauges, ultrasound-responsive bioinks were able to be bioprinted with high feature fidelity. Thus, it allows bioprinting filaments with the desired size and printing properties, which is important for bioprinting complex tissue-mimicking structures which are comprised of features with varying size requirements. An example of this is the utilization of ultrasound-responsive gene-delivery microbubbles in small, detailed prints with finer features that mimic native tissues and mitigate the limitations of nutrient diffusion, while maintaining the ultrasound-mediated spatiotemporal control of gene delivery. This technology could be used to model early-stage diseases where only a small subset of cells begins to express a target gene. For example, ultrasound-responsive targeted gene delivery of oncogenes to nearby epithelial cells could be used to assess cancer’s interactions with the environment and crosstalk with neighboring cell types such as endothelial or stromal cells. The knowledge of how to control feature sizes during the bioprinting process will enable different cellular arrangements and architectures to be studied in this system. This technology also leverages these microbubbles with focused ultrasound to overcome diffusion barriers presented by the matrix material itself and enable user-defined genetic manipulation with spatiotemporal control in high-fidelity bioprinted constructs. Using optimized parameters, the ability to locally manipulate cells in bioprinted constructs using focused ultrasound was shown in embodiments. These established parameters enable user-defined remote-controlled genetic manipulation in bioprinted constructs that can be leveraged to identify drivers in early disease and could be used to remotely mediate gene expressions for applications in tissue regeneration. (III) Three-dimensional bioprinting technique / method to preserve microbubble stability and embedding in the cast collagen gel [000142] FRESH (Freeform Reversible Embedding of a Suspended Hydrogel) 3D bioprinting using a CellInk BioX6 printer with pneumatic print heads printing through a 22G conical needle. Printing uses an ink comprising 4-8% sodium alginate, HEK293T (human embryonic kidney) single cells, or MCF10A human breast epithelial spheroids (produced using a U-Bottom well plate culture technique), and ultrasound-responsive microbubbles coupled with DNA plasmids. Gelatin microparticle support bath beads are resuspended in cell media that has an additional 0.2% calcium chloride added to it. The calcium chloride is necessary for crosslinking the alginate bioink, and the presence of cell media promotes the viability of the cells without hindering the support properties of the bath. The support bath mixture is added to an 8-well LabTek 1.0 coverglass-bottom well. Ink containing the gene delivery microbubbles and cells isprinted into the support bath using the Freeform Reversible Embedded Suspension of Hydrogels (FRESH) method. Upon completion of printing, cell media with an additional 0.2% calcium chloride is poured over the support bath that contains the bioprint. This is then placed into an incubator for 15-20 minutes to dissolve the support bath material. The dissolved support bath material is removed from the printed construct, and the construct is then rinsed with cell media and suspended in fresh cell media. [000143] Coaxial extrusion 3D bioprinting using CellInk BioX6 printer with pneumatic print heads through a coaxial (16G OD (outer diameter), 20G ID (inner diameter)) bioprinting needle. Printing uses an inner “core” ink comprising 4-8% sodium alginate, HEK293T (human embryonic kidney) single cells, hFOB 1.19 human osteoblasts, or MCF10A human breast epithelial spheroids (produced using a U-Bottom well plate culture technique), and ultrasound- responsive microbubbles coupled with DNA plasmids. The outer “shell” ink is 2% gelatin mixed with 2% calcium chloride to facilitate the crosslinking of the alginate immediately upon being printed. [000144] For both printing methods, after the prints are crosslinked and removed from their respective printing mediums, a solution of 8 mg / mL Type I collagen, working solution (71.55% 1X +Ca / +Mg DPBS, 25.00% 10X +Ca / +Mg DPBS, 3.45% NaOH), and cell media are added to the well containing the bioprint. The print is left at room temperature for 10 minutes, then placed into the incubator for 45 minutes to complete crosslinking. Media is added to the top of the collagen before exposure to ultrasound. The ultrasound transducer is focused to have the ultrasound focal zone in the middle of the bioprinted filaments. The construct is then exposed to ultrasound at a frequency of 2.25 MHz, 10 ms pulse, 1 ultrasound pulse per second, 10-80 pulses. After 48 hours, the cells within the ultrasound focal zone begin to overexpress the plasmid that was coupled to the microbubbles. [000145] While both the coaxial and freeform bioprinting approaches achieve ultrasound- localized transfection, there are many additional and more complex capabilities that the freeform approach enables, as shown in the exemplary embodiments. The freeform technique is able to produce intricate hollow perfusable networks, including branched vessels, which are highly relevant for printing vascular networks seen in real tissue. Additionally, freeform approaches are better suited to creating entire volumetric or organ-like structures, such as multi-chamber heart structures or a heart valve. Additional structures that can be printed with the freeform approach include spiral and nested configurations, interlocking rings, and complex pore networks. The freeform approach is also better suited to printing multicellular spheroids or organoids (as demonstrated in the exemplary embodiments), as this technique is more amenable tomaintaining high viability of these multicellular units during printing. Further, by using the dissolvable support bath, the freeform approach can print structures that would otherwise collapse during printing such as structures with overhangs as well as linked rings and dumbbells. These are all unique geometries that can impart different mechanical properties on the tissue and can affect the state and architecture of the cells grown in or on these printed structures. [000146] In embodiments herein, the hollow bifurcated structure shown in freeform bioprinting approach is a highly relevant geometry for printing perfusable branched tissue vasculature, which is not feasible to achieve by printing planar filament structures of coaxial bioprinting. The hollow bifurcated structure solves the issue of creating hollow branched tissue structures that are seen in vasculature and ductal tissues (e.g. mammary glands, bronchial tree of the lung). (IV) Kits [000147] In another embodiment, the present invention contemplates kits for the practice of the methods of this invention. [000148] The kits preferably include one or more containers containing various components to practice at least one embodiment of this invention. The kit can optionally include a first container comprising an ultrasound targeted microbubble population. The kit can optionally include a second container comprising a plurality of nucleic acids to be expressed. The kit can optionally include a third container comprising a plurality of mammalian cells or cell aggregates. The kit can include a fourth container comprising a printable bioink (e.g., hydrogel). The reagents may be provided together in a suspension or may be provided as separate components which can be combined later. The kit can further include a fifth container comprising collagen (e.g., type I, type II, type III and the like). The kit may optionally contain additional agents with the microbubble population. The nucleic acids in the second container may include, without limitation, oligonucleotide molecules, siRNA, shRNA, RNAi, miRNA, transcription factor decoy molecules, deoxyribonucleic acid vectors, DNA, plasmid DNA, genes, and gene fragments. For example, the plasmid DNA may be a pCAG-GFP plasmid. Other nucleic acid molecules may include DNA expressing VEGF or HER2 cancer genes, for example. The microbubble population in the first container may comprise a 1-5 μm sized cationic ultrasound-responsive microbubbles for gene delivery. The mammalian cells or cell aggregates in the third container may comprise HEK293T cells, osteoblast cells, breast epithelial spheroids, or other cell types. The printable bioink in the fourth container may comprise alginate, gelatin, GelMA, or other hydrogels. The collagen in the fifth container may comprise type I collagen or other variations ofcollagen. The kits may also optionally include appropriate systems (e.g. opaque containers) or stabilizers (e.g. antioxidants) to prevent degradation of the reagents by light or other adverse conditions. [000149] The kits may optionally include instructional materials containing directions (i.e., protocols) providing for the use of the reagents in the methods of stably binding nucleic acids to an ultrasound-responsive microbubble population. Further, the instructions may include providing for the use of the reagents in the methods for integrating microparticle-coupled nucleic acid molecules with a plurality of mammalian cells / cell aggregates in a collagen encased three- dimensional bioprinted matrix. Furthermore, the instructions may include providing methods for the use of various ultrasonic parameters (e.g., frequency, number of pulses, pulse duration, and the like) for a focused ultrasound exposure at a desired location of the matrix for localized transfection of cells. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials. [000150] The examples below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the scope of the disclosure. (V) Examples [000151] Example 1: Materials and Methods [000152] Fabricating ultrasound-responsive microbubbles: Cationic ultrasound-responsive microbubbles (μB) were utilized to facilitate ultrasound-responsive gene delivery within the disclosed bioprinted structures. These microbubble particles contain a gas core of a mixture of air and low-solubility perfluorocarbon gas, stabilized by a lipid monolayer. The cationic lipid surface of the microbubble allows for electrostatic coupling of plasmid DNA.9,10,43,53,54The microbubbles were fabricated prior to incorporation within bioinks. Briefly, the lipids 18:0 PC (1,2-distearoyl-sn-glycero-3-phosphocholine, 27.2g, Avanti Polar Lipids), 18:0 TAP (1,2- stearoyl-3-trimethylammonium-propane (chloride salt), 6.4g, Avanti Polar Lipids), and 18:0 PEG2000 PE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt), 6.4g, Avanti Polar Lipids) in powder form were combined in aglass scintillation vial and suspended in 1X DPBS without calcium or magnesium (-Ca / -Mg, 6mL, Gibco). The solution was sonicated using a 3.4mm sonication probe for 3 minutes at 90% intensity to thoroughly mix the lipids. Upon mixing, hydrophobic perfluorocarbon (PFC) gas (octafluoropropane, APF-N40HP (99.8%), FluoroMed) was sparged into the scintillation vial and sonicated at 90% intensity for 10 seconds, resulting in the formation of microbubbles with a PFC and air gas interior and lipid monolayer exterior including the cationic 18:0 TAP lipid. The microbubbles were rested at room temperature for 15 minutes and then added to a capped glass vial and centrifuged at 1500 RPM for 1 minute. This step caused the microbubbles to float and separate from the subnatant of DPBS buffer and excess lipid. This subnatant was then removed using a syringe with a 22G needle, and 2mL of fresh 1X DPBS was added to the vial before tapping to mix and centrifuging again. This process was repeated for a total of 5 washes to remove excess lipids. The microbubbles were then resuspended in 300μL of 1X DPBS, tapped to mix well, and counted to obtain a concentration of μB / mL using a hemocytometer and brightfield microscope. [000153] Coupling pCAG-GFP DNA plasmid to ultrasound-responsive microbubbles: pCAG-GFP plasmid was added at a ratio of 0.1 pg of pCAG-GFP plasmid DNA per microbubble. pCAG-GFP was a gift from Connie Cepko (Addgene plasmid # 11150 ;http: / / n2t.net / addgene:11150 ; RRID:Addgene_11150).38After adding the required solution ofplasmid DNA, the vial containing the microbubbles was shaken to thoroughly mix the plasmid and microbubble solution, and was mixed every 15 minutes for two hours. After the microbubbles were coupled with DNA, they were counted again via hemocytometer and brightfield microscope to determine a final concentration of the stock microbubble solution prior to incorporation into the bioinks. For visualization of coupled DNA, plasmid-coupled microbubbles were stained with YOYO-1 Iodide nucleic acid stain (Thermo Fisher Scientific) and washed with 1X DPBS prior to imaging. Overlaying brightfield and the fluorescence images of the microbubbles, allows us to visualize the DNA coupling, as fluorescent signal of the YOYO-1-stained DNA localizes to the surface of the microbubbles. [000154] Size distribution of ultrasound-responsive microbubbles: The ultrasound- responsive microbubbles were diluted at a factor of 1:200 and placed between two 1.5 glass coverslips before imaging on the Leica Thunder Microscope. Microbubble imaging was performed using a 63X oil immersion lens. Microbubble size analysis was obtained by automated particle analysis performed in ImageJ. Briefly, image thresholding was applied to segment the microbubble particles and the automated fill holes and watershed functions were applied to the thresholded microbubble image. Automated particle sizing was performed on thesegmented microbubbles by specifying a minimum pixel size and selecting a 0.8-1.0 circularity interval. [000155] Fabricating bioinks for bioprinting constructs: Sodium alginate is a naturally occurring polymer that is non-toxic and biodegradable, with a tunable range of mechanical properties.26,27,56Alginate is frequently utilized as a bioink3and concentrations of 2-6% w / v have been previously employed to simulate tissue environments.7,27,36,62Alginate bioinks were prepared at 2% w / v, 4% w / v, and 6% w / v by adding powdered sodium alginate (3D Systems) to DI water (UltraPure™ DNase / RNase-Free Distilled Water, Invitrogen). These inks were vortexed to mix and then added to a bead bath at 60˚C for 15 minutes. The inks were vortexed again and stirred using a metal stirrer to break up large powder fragments before being placed in the bead bath for another 15 minutes. This process was repeated a minimum of 3 times, or until the alginate powder and water completely incorporated together. The alginate bioinks were then centrifuged at 1000RPM for 5 minutes to remove any air bubbles that were incorporated during the mixing process. Alginate functionalized with RGD peptide (arginylglycylaspartic acid), which promotes cellular adhesion within the bioprint,6was prepared for printing the osteoblasts by diluting 10% w / v A-RGD (CellInk) with cell media for a final concentration of 4% w / v alginate. Gelatin bioink with calcium chloride crosslinking agent was prepared by adding gelatin (gelatin from bovine skin Type B, Sigma Aldrich) to a solution of calcium chloride (CaCl2, 3D Systems) in DI water for a final concentration of 2% w / v gelatin, 2% w / v CaCl2. Gelatin is used to increase the viscosity of the crosslinker bioink, as it is challenging to control the flow of less viscous liquids when extrusion bioprinting. After crosslinking the alginate, the gelatin was dissolved by placing the print at 37˚C and was removed by rinsing with fresh media. The ink was vortexed to mix and added to a bead bath at 85˚C for 5 minutes. The solution was vortexed again and stirred using a metal stirrer to break up large powder fragments before being placed back into the 85˚C bead bath for an additional 25 minutes. The ink was placed at 4˚C for 15 minutes, then placed in the bead bath at 85˚C for 30 minutes. This process was repeated for 3 total rounds of heating and cooling. After the final cycle, 2mL of the gelatin bioink was pipetted into a 3mL bioprinting syringe (CellInk). The solution was allowed to cool at room temperature overnight to partially solidify the gelatin for bioprinting ease. [000156] Forming breast epithelial spheroids: Breast epithelial spheroids were formed by pipetting 200μL of MCF10A cells (MCF10A ATCC CRL-10317) between P3-P10 in complete MCF10A media containing DMEM / F12 (Invitrogen) supplemented with 5% heat activated horse serum (Invitrogen), epidermal growth factor (EGF) (20ng / μL, Peprotech), hydrocortisone (0.5ng / μL, Sigma), cholera toxin (100ng / μL, Sigma), insulin (10μg / μL, Sigma), and 1% pen-strep (penicillin-streptomycin, 10,000 μg / mL, Gibco) into 96-well low-adhesion U-bottom plates (Nunclon Sphera 96-UWell Round Bottom Plate, 300μL well volume, Thermo Scientific) at a concentration of 15,000 cells / mL. The plates were then centrifuged for 5 minutes at 400 RPM in a swinging bucket rotor centrifuge. Then plates were rotated 180 degrees and then centrifuged again for 3 minutes at 400 RPM. The two separate acceleration periods were found to promote circular spheroid formation. The cell clusters were cultured in the incubator for 4 days, and spheroids formed over this period. [000157] Incorporating plasmid-coupled ultrasound-responsive microbubbles into alginate bioinks: Alginate bioinks were prepared as previously stated, and 400μL of the respective bioink was added to a 3mL bioprinting syringe (CellInk). Plasmid-coupled microbubbles were prepared as previously stated, and added to the 2%, 4%, and 6% w / v alginate bioinks at a final concentration of 2.34x109μB / mL and stirred gently to avoid the incorporation of air bubbles using a 22G blunt-tip needle attached to a syringe. [000158] For HEK293T printing, microbubbles coupled with respective plasmids were prepared as stated and added to the alginate bioinks at a final concentration of 2.34x109μB / mL with HEK293T cells at a final concentration of 3.85 x 106cell / mL and stirred gently to mix with a blunt-end 22G needle, which reduced the formation of air bubbles. [000159] Breast epithelial spheroids (MCF10A) were pre-coupled to the microbubbles to ensure close contact between the microbubbles and spheroids, which aids in ultrasound-responsive gene delivery. Breast epithelial spheroids were pipetted into a 50mL conical tube for a final bioink concentration of 1.44 x 103spheroid / mL. Excess media was aspirated leaving only the spheroids. Plasmid-coupled microbubbles were added the spheroids for a final bioink concentration of 2.34x109μB / mL, and gently hand-flicked to mix. The solution was placed into the incubator at 37°C and mixed every 2 minutes for 10 minutes. After mixing, the spheroids pre-coupled with microbubbles were pipetted into the alginate in the bioprinting syringe, mixed gently with a blunt 22G needle, and immediately loaded into the bioprinter at 37°C. [000160] Incorporating cells into microbubble-laden alginate bioinks: HEK293T (ATCC CRL-3216) cells were obtained at 80% confluency between P6-P16 from a T75 flask using TrypLE (TrypLE™ Express, Gibco) to lift them. They were then suspended in complete media (DMEM, high glucose, Gibco), 10% FBS (HyClone Characterized Fetal Bovine Serum CA Origin, Cytiva), and 1% pen-strep (penicillin-streptomycin, 10,000 μg / mL, Gibco) and concentrated to 3.85 x 107cell / mL in complete media. hFOB 1.19 (ATCC CRL-3602) cells were obtained at 80% confluency at P12 from a T75 flask using TrypLE (TrypLE™ Express, Gibco) to lift them. They were then suspended in complete media containing Human Osteoblast Media(Cell Applications, Inc.), 10% FBS (HyClone Characterized Fetal Bovine Serum CA Origin, Cytiva), and 1% pen-strep (penicillin-streptomycin, 10,000 μg / mL, Gibco) then concentrated to 3.85 x 107cell / mL in complete media. The CellInk Bio-X6 bioprinter was set to 37˚C to maintain viability of the cells. Upon preparation for bioprinting, 20μL of the appropriate cells at 3.85x107cell / mL were added to the syringe containing the previously prepared solution of alginate bioink and ultrasound-responsive microbubbles. The cells were mixed in by stirring gently to avoid the incorporation of air bubbles using a 22G blunt-tip needle attached to a syringe. The loaded bioprinting syringe was then immediately loaded into the bioprinter at 37˚C. [000161] Coaxial bioprinting of cell-laden ultrasound-responsive alginate bioinks: The CellInk Bio-X6 bioprinter with DNA Studio 4.0 software was used throughout all experiments. The previously prepared syringe containing the cell-laden ultrasound-responsive alginate bioink was attached to the core inlet of a coaxial nozzle at 37˚C, and the previously prepared syringe containing the 2% gelatin in 2% CaCl2was attached to the sheath inlet of the coaxial nozzle (20G inner diameter core, 16G outer diameter sheath (3D Systems)) at room temperature. The bioprinter was calibrated to touch the top of a glass slide and then moved upward 0.2mm to give clearance space for the print to extrude. An 8-well dish (LabTek 8-well 1.0 cover glass bottom, LabTek) was needed for ultrasound manipulation of the bioprinted construct but caused a lack of clearance with the printing needle. This meant the construct was printed directly onto a slide and the slide was transferred to the 8-well dish after printing. The bioinks were printed at 4mm / s, with the alginate printing at 37˚C (for cell viability) in the range of 10-15 kPa, and the gelatin printing at room temperature in the range of 40-60 kPa. A pre-flow delay of -400ms and a post-flow delay of -400ms was used for all inks to aid print fidelity. The pre-flow delay allowed for the bioink to begin extruding briefly before the printer nozzle began moving, which aided the ability of the ink to be printed onto the slide without dragging the filament around. The post-flow delay ensured that the printing pressure was removed before completing the print to prevent smearing and account for nozzle leakage. Both of the flow delays were negative values, meaning that the pre-flow delay caused the ink to extrude before the nozzle began moving, and the post-flow delay caused the ink to stop extruding before the nozzle stopped. The alginate core containing the microbubbles and cells was surrounded by the 2% gelatin 2% CaCl2sheath enabling the alginate to crosslink upon exiting the coaxial nozzle. [000162] Preparing support bath for extrusion bioprinting: Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting utilizes a support bath containing a slurry of gelatin beads called LifeSupport® to allow bioprinting of freestanding multilayer volumetric geometries. Sodium alginate can be crosslinked immediately upon printing via contact withcalcium chloride; as such, the support bath contains calcium chloride to facilitate this crosslinking. To prepare the support bath, calcium chloride (CaCl2) (40mg, 3D Systems) is added at a concentration of 0.2% CaCl2to 20mL of the respective complete media for either HEK293T cells or MCF10A spheroids at 4°C. The media is mixed thoroughly to incorporate the CaCl2, then added to a 50mL conical tube containing LifeSupport® (500mg, Sigma Aldrich) and vortexed for 1 minute to ensure the support slurry is saturated with media. The tube containing the slurry is placed at 4°C for 15 minutes to allow for full absorption of media, after which the solution is centrifuged for 7 minutes at 2100 RCF at room temperature. The centrifugation forms a compacted subnatant of the support bath, and a supernatant of excess media, which is removed. Before bioprinting, 300μL of the support slurry is added to a corner well of an 8-well dish (LabTek 8-well 1.0 coverglass bottom, LabTek). [000163] FRESH 3D bioprinting ultrasound responsive bioinks containing cells or spheroids: The CellInk Bio-X6 bioprinter equipped with DNA Studio 4.0 software was used for all bioprinting experiments. Once prepared as previously stated, the 3mL syringe with a 22G conical nozzle containing 4% w / v alginate bioinks laden with plasmid-coupled ultrasound responsive microbubbles at 2.34 x 109μB / mL and either HEK293T cells (3.85 x 106cell / mL) or MCF10A spheroids (1.44 x 103spheroid / mL) was loaded into the bioprinter at 37°C. Prints containing spheroids utilized 4% w / v RGD-alginate (A-RGD, Cellink) to promote cellular adhesion. The bioprinter was calibrated to touch the tip of the nozzle to the glass coverslip of the center of a corner well of the LabTek 8-well dish containing the LifeSupport® slurry, then moved upward 0.2mm to give clearance for the bioink to extrude from the nozzle. After calibration, the nozzle is wiped off to remove any excess slurry that could prematurely crosslink the alginate. [000164] The bioinks were printed at 37°C to improve cellular viability. To print single filaments, a print speed of 4mm / s was used, and the range of pressures to print the bioinks ranged from 10-14 kPa. For 2x2 lattice prints, a 25G nozzle was used with a pressure of 16-20 kPa. For all prints, a pre-flow delay and post-flow delay of -400ms was used to aid print fidelity. The negative pre-flow delay causes pressure to be applied to the syringe before the printhead moves, allowing the ink to begin printing in the desired location without dragging the filament. The post-flow delay causes the pressure to stop just before the print is finished which prevents smearing and accounts for nozzle leakage. The ink was printed into the support slurry containing calcium chloride, causing the alginate to crosslink upon contact with the slurry. [000165] To print constructs containing microbubbles for expression of multiple genes, two different bioinks were used. Bioinks of 4% w / v alginate containing HEK293T cells andmicrobubbles were prepared as previously described. Two different inks were loaded into the bioprinter: one containing microbubbles pre-coupled with plasmid DNA to cause the expression of GFP, and the other ink containing microbubbles pre-coupled with plasmid DNA to cause the expression of mCherry. These filaments were printed side by side into a 2-well dish (LabTek 2- well 1.0 coverglass bottom, LabTek) containing 2mL of support slurry in an alternating pattern at 10-14 kPa with a speed of 4mm / s and using a pre and post flow delay of -400ms. Upon contacting the calcium chloride support slurry, the alginate bioink begins to crosslink; when another layer is immediately printed in contact with the first layer, partial crosslinking of the alginate filaments occurs to create a single multilayer construct. This concept is also applied when printing bottom-up multilayer complex structures, such as a bifurcated vessel into a 2-well LabTek dish containing 2mL of support slurry. A 4% w / v alginate bioink is prepared with microbubbles pre-coupled to plasmid for GFP expression at 2.34 x 109μB / mL with HEK293T cells as previously described. The bifurcated construct was printed at 3mm / s with pressure varying from 11-17kPa using a 22G blunt-tip nozzle. For this multilayer construct, each layer is printed on top of one another, causing some crosslinking between the layers to form a single bioprinted construct. [000166] Upon printing completion, the LifeSupport® slurry was topped with respective cell media containing 0.2% CaCl2 at 37°C, and the entire dish was placed at 37°C for 5 minutes. This dissolves the slurry and leaves a freestanding 3D printed structure. The dissolved support bath and media were aspirated, and 400μL of respective new media was added to the dish before placing it back at 37°C for 5 minutes to remove any excess LifeSupport® or calcium chloride. This media is then aspirated before continuing. [000167] Gene delivery in cell-laden ultrasound-responsive alginate bioinks: Once the cell-laden ultrasound-responsive constructs had been printed, 400μL of respective complete media was added to each of the bioprinted constructs and they were placed into an incubator at 37˚C, 5% CO2for 15 minutes to dissolve gelatin on the outside of the constructs. The media was changed once to completely remove the excess gelatin. The media was removed from the bioprints before continuing. To facilitate the use of focused ultrasound, 250 μL of 2.7 mg / mL type I collagen solution (Collagen from rat tail tendon, Roche) in acetic acid that had been pH neutralized with NaOH to an isotonic solution was pipetted on top of each construct. The dish was then rocked gently to ensure even distribution of the collagen. Next, the dish was placed into an incubator at 37˚C, 5% CO2for 45 minutes, polymerizing the collagen. Upon collagen casting, the bioprinted filaments were completely embedded in the gelled collagen, which facilitated the use of focused ultrasound by holding the filament in place and minimizinginterface density differences that can scatter the focused ultrasound beam. After the collagen had gelled, 400μL of the cell-appropriate complete media was added to each construct- containing well. The bioprinted constructs were then loaded into a custom ultrasound / optical microscope setup consisting of a 2.25 MHz spherically-focused ultrasound transducer (Olympus V305-SU) mounted in a tank of DI water, a robotic arm to position the sample, and a Nikon microscope body with high-speed camera (Photometrics Prime 95B sCMOS) attached to a 4X air objective lens (Nikon). The ultrasound focal zone was colocalized with the optical focal zone of the microscope objective. The ultrasound transducer was driven by a Class AB ultrasonic amplifier (Vox Technologies) and an arbitrary waveform generator (National Instruments PCI- 5412). LabView 2016 software (National Instruments) was used to integrate the transducer, camera, and robotic arm for sample movement. Next, the ultrasound was focused 250 μm deep into the construct and a 10 ms focused ultrasound pulse (2.25 MHz, approximately 1.8 MPa peak negative pressure) was applied for 10, 40, or 80 pulses at 1 second intervals. The constructs were then placed back into the incubator at 37˚C, 5% CO2for 48 hours before assessing gene expression with a media change at 24 hours. [000168] FRESH 3D bioprinting of hollow bifurcated constructs with ultrasound responsive bioinks containing cells: Alginate bioinks were prepared as previously stated with 4% w / v alginate, HEK293T cells at 3.85 x 106cell / mL, and GFP-coupled microbubbles at 2.34 x 109μB / mL. A 24-well plate was filled with 1.5mL of LifeSupport® slurry containing 0.2% CaCl2. The bifurcated construct was printed upright to ensure hollow tube formation; a 25G blunt-tip needle was used with a pressure of 16-20kPa, with printing speed of 2mm / s, and a pre-flow delay of -400ms. Upon printing completion, the construct was allowed to crosslink at room temperature for 5 minutes. The well plate was then moved to the tissue culture hood and turned sideways before removing the print and surrounding LifeSupport® with a spatula. The construct was placed onto the bottom glass of a 2-well LabTek dish that had the walls removed. A 20G blunt needle was inserted into cavity of the central tube, with a 25G blunt needle inserted into each cavity of the bifurcated tube ends. The construct was then covered in 1mL of HEK293T media and placed into the incubator at 37°C for 10 minutes. Upon support bath dissolution, the media and liquefied support bath were gently aspirated, and an additional 1mL of media is added before returning the construct to the incubator for 5 minutes. This media is then aspirated, and the walls of the 2-well LabTek dish are carefully reattached to the bottom coverslip around the construct. Neutralized collagen is prepared as previously stated and 1mL is added to the well containing the bioprinted construct before returning to the incubator for 1.5 hours to initially crosslink the collagen. After crosslinking, the walls are removed leaving acollagen puck containing the bioprinted construct and needles. The needles are gently removed using a magnetized pair of tweezers. The collagen puck containing the construct are then transferred to an intact 2-well LabTek dish and topped with approximately 750μL of neutralized collagen, then placed back into the incubator for 45 minutes.1mL of media is gently added to the top of the print before returning to the incubator for 2 hours to allow the collagen to have completely crosslinked. Focused ultrasound may then be applied as previously described, with 40 pulses applied to the larger tube or 25 pulses in one of the smaller tubes. After ultrasound application, an additional 1mL is added to the well. Media is changed every 24 hours with 2mL of new media. [000169] Fixing and staining cell-laden constructs for gene delivery visualization: 48 hours after ultrasound exposure, cell-laden constructs were fixed for 45 minutes at room temperature with 4% paraformaldehyde (Pierce™ 16% Formaldehyde (w / v), Methanol-free, Thermo Scientific) in +Ca / +Mg 1X DPBS (Gibco). Each sample was rinsed three times with 400μL of +Ca / +Mg 1X DPBS. Samples were then permeabilized for 30 minutes at room temperature with 0.2% Triton X-100 (Thermo Scientific) in +Ca / +Mg 1X DPBS. The solution was removed from the constructs and 32.4μM Hoechst nuclear stain (Hoechst 33342, Trihydrochloride, Trihydrate, Invitrogen) in 0.2% Triton X-100, +Ca / +Mg 1X DPBS was added to each construct for 1 hour at room temperature. After staining, each sample was washed 3 times for 15 minutes in the incubator at 37˚C, 5% CO2with +Ca / +Mg 1X DPBS before imaging. Bifurcated prints were washed overnight. [000170] Brightfield and fluorescence microscopy and image analysis: Fluorescence microscopy was performed using the Leica Thunder 3D cell culture widefield microscope imaging system. Samples were imaged in brightfield and fluorescence with three replicate samples per condition. Each sample was imaged while maintaining the same image acquisition settings including exposure time and laser intensity across all samples within experiments. Image analysis for quantification of the microbubble activation zone within filaments and microbubble stability were performed using ImageJ as described in the sections below. Prior to measurements and analysis, the image size scale was calibrated in ImageJ using image scale information from the calibrated microscope. The analyses of transfection efficiency and cell viability were performed using Volocity Image Processing Software. [000171] Analysis of bioprinted microbubble stability over time: To determine the stability of bioprinted microbubbles over time, filaments of 4% w / v sodium alginate containing HEK293T cells and 2.34x109μB / mL were bioprinted as previously described. Immediately post-printing, the constructs were cast in collagen and allowed to crosslink in the incubator as describedabove. After adding 400μL of complete media to each well, dishes containing the filaments were sparged with octofluoropropane gas and parafilmed to promote stability. Room temperature (RT) samples were left on the benchtop, while incubated samples (37°C) were placed into the incubator. Each timepoint and temperature condition had 3 replicates. At each timepoint, (0 hr, 1 hr, 3 hr, 6 hr, 12 hr, 24 hr, 36 hr, and 48 hr post-printing) samples were imaged using brightfield microscopy on the Leica Thunder widefield microscope. Microbubble stability was determined by measuring transmitted light through the filament at set time points. If microbubbles destabilized, they would no longer scatter transmitted light and more transmitted light would pass through the filament. To assess stability by image analysis, using ImageJ, a circular ROI 500μm in diameter was placed within the filaments, and the average pixel intensity was calculated and averaged among 3 samples for each condition. The baseline of 100% intensity was established for samples at 0 hr, to which all other samples were compared. For each timepoint, normalization was done by dividing the average 0 hr pixel intensity by the average pixel intensity of that timepoint and multiplying by 100 to obtain a percentage. [000172] Performing rheological studies on ultrasound responsive bioinks: Shear rate sweeps were conducted using a TA Instruments Discovery HR-1 rheometer equipped with a peltier plate stage and a 40mm parallel plate geometry. Approximately 1mL of acellular alginate at varying concentrations (2% w / v, 4% w / v, and 6% w / v) both containing no microbubbles or containing microbubbles at 2.34 x 109μB / mL were individually loaded onto the stage. The size gap on the instrument was set to 650μm, and any excess bioink was wiped away. Shear rate sweeps were performed at a range of 10-1000s-1at room temperature. There were three replicates for each condition (N=3). [000173] Analyzing size of the visual activation zone of microbubbles post-ultrasound: Bioinks consisting of 4% w / v sodium alginate containing 1.56x109μB / mL, 2.34x109μB / mL, and 3.51x109μB / mL were prepared, bioprinted, and prepared for ultrasound exposure as previously described (N=3 replicate samples per condition). All samples were then imaged using brightfield microscopy on the Leica Thunder widefield microscope. After imaging, the filaments were individually exposed to 40 pulses of focused ultrasound, focused on the top plane of the filament, and imaged again post-ultrasound. Using ImageJ, a line, centered to the filament, was drawn along the length of the filament and a pixel intensity plot along the line was generated. Microbubbles scatter transmitted light, therefore regions containing more microbubbles have lower pixel intensities than regions with fewer microbubbles. The pixel intensity for the top 200μm of the line plot was averaged to define a baseline and compared to intensity values further down the filament. The top edge of the ultrasound activation zone was defined as wherethe value on the line profile exceeded three times the average baseline intensity value. This analysis was repeated for the bottom edge of the activation zone using the bottom 200μm of the line plot to define the lower baseline value. The distance between the top and bottom edges was measured and reported as the diameter of the activation zone. [000174] Analysis of cell viability in alginate bioprinted constructs: Bioinks of 2%, 4%, and 6% w / v alginate containing HEK293T cells and 2.34x109μB / mL were prepared and printed as previously described. At either 0 hr post-printing or 48 hr post-printing, the filaments were stained with calcein-AM (LIVE / DEAD™ Viability / Cytotoxicity Kit, for mammalian cells, 8μM, ThermoFisher) and ethidium homodimer-1 (LIVE / DEAD™ Viability / Cytotoxicity Kit, for mammalian cells, 4μM, ThermoFisher) diluted in complete media (DMEM, 10% FBS, 1% pen- strep) for 45 minutes in the incubator. Samples stained at 48 hr post-printing were given media changes daily prior to staining. After staining as aforementioned, the filaments were washed with complete media 3 times for 5 minutes each in the incubator. The filaments were then imaged using fluorescence microscopy on a Leica Thunder widefield microscope. To determine viability, Volocity Image Processing software was used to segment and count the number of live and dead cells within each filament. Viability was calculated by dividing the number of live cells by the number of total cells (live + dead), then multiplying by 100 to obtain a percentage. Each condition had three replicate samples. [000175] Analysis of ultrasound pulse number effect on cell transfection: Bioinks containing 4% w / v alginate with HEK293T cells and 2.34x109μB / mL with coupled GFP plasmid were bioprinted and prepared for ultrasound exposure as previously described. Samples were imaged using brightfield microscopy before ultrasound exposure. Each sample was then exposed to 10, 40, or 80 ultrasound pulses focused at a depth of 250 μm into the filament (N=3 replicate samples per condition) and imaged again in brightfield. The samples were then placed back into the incubator with daily media changes until the 48 hr post-ultrasound timepoint. Control samples exposed to 0 ultrasound pulses (N = 3 replicate samples) were prepared with the same protocol. All samples were fixed and stained with Hoechst nuclear stain as previously described before imaging using brightfield and fluorescence microscopy on the Leica Thunder widefield microscope. Volocity Image processing software was used to segment and count the number of GFP transfected cells in each filament. To determine the diameter of the zone containing transfected cells, ImageJ was used to draw a straight vertical line parallel to the two transfected cells furthest from each other in each bioprinted sample. This line was then measured and reported as the diameter of the zone containing transfected cells.[000176] Analysis of microbubble concentration effect on transfected cells: Bioinks containing 4% w / v sodium alginate with HEK293T cells and 7.81x108μB / mL, 1.56x109μB / mL, 2.34x109μB / mL, and 3.51x109μB / mL were prepared, bioprinted, and prepared for ultrasound exposure as previously described. All samples were then imaged before ultrasound using brightfield microscopy. Each sample was then exposed 40 ultrasound pulses focused at a depth of 250 μm into the filament (N=3 replicate samples per condition) and imaged again in brightfield. The samples were then placed back into the incubator with daily media changes until the 48 hr post-ultrasound timepoint. Control samples containing 2.34x109μB / mL with no plasmid DNA, and containing no microbubbles and no DNA were prepared and exposed to ultrasound with the same protocol (N=3 replicate samples per condition). All samples were fixed and stained with Hoechst nuclear stain as previously described before imaging using brightfield and fluorescence microscopy on the Leica Thunder widefield microscope. Volocity Image processing software was used to segment and count the number of GFP transfected cells in each filament. To determine the diameter of the zone containing transfected cells, ImageJ was used to draw a straight vertical line parallel to the two transfected cells furthest from each other in each bioprint as described in the previous section. [000177] Statistical Analysis: All quantitative experiments were performed with a minimum of N=3 replicates. Unless otherwise noted, the data presented are means + / - standard deviation. All statistical analyses were performed using GraphPad Prism 10 Software. One-way analysis of variance (ANOVA) with Tukey’s post hoc multiple comparisons tests were performed on each measurement with multiple groups compared. p values of <0.05 were considered significant and denoted with * (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [000178] Determining optimal bioprinting pressure and speed: To determine optimal bioprinting parameters for each printing needle gauge, (blunt-tip 1” needle: 20G, 22G, 25G, 27G, 30G, CellInk) the extrusion pressure was increased until the bioink began to flow from the print needle tip. This was the initial starting point for determining optimal printing pressure. Filaments were bioprinted into the support bath beginning at this starting pressure and increasing in pressure until the target filament diameter (based on the nominal inner diameter (I.D.) of the print needle) was achieved. Once an optimal pressure was determined, the filaments were printed in triplicate at the optimal pressure, as well as 2 kPa above and below optimal with and without microbubbles for comparison. The optimal bioprinting pressures for each print needle gauge are listed in Table 1. [000179] Determining optimal bioprinting speed: The bioprinting speed was determined using optimal bioprinting pressures that were characterized for each print needle gauge. Thefastest speed that results in optimal filament diameter is preferred, as it reduces the time to create printed constructs, increases cellular viability by reducing time out of the incubator, and promotes the crosslinking of layers with each other in multi-layer bioprints. Initially, all print needle gauges were printed at 4 mm / s, as this was determined to be optimal for a 20G nozzle. However, it was determined that print needle gauges above 25G must be printed at lower speeds to achieve target filament diameter. The filaments were bioprinted into the support bath at their optimal pressures and optimal print speed determined. Once the parameters were set, the filaments were printed in triplicate at the optimal printing pressure and speed, as well as 1 mm / s above and below the optimal speed with and without microbubbles for comparison. The optimal bioprinting speeds for each print needle gauge are listed in Table 1. [000180] Characterization of bioprinted filament diameter: The bioprinted filaments containing microbubbles were bioprinted as described and imaged in brightfield and fluorescence microscopy on the Leica Thunder microscope with three replicates for each condition. To determine the diameter of the bioprinted filament, ImageJ v1.54p was used on these microscopy images. Briefly, the scale was calibrated and each bioprinted filament was analyzed similarly for each replicate across all conditions. The images were adjusted to 8-bit and a threshold was set similarly for each needle gauge, and set to make the fluorescence image binary, where the outline of the filament was separated from the background. The same threshold was used for each needle gauge, with and without microbubbles across replicates. Using the “analyze particles” function, the area was measured for the bioprinted filament. The length of the filament analyzed was measured, and the area was divided by this value to calculate an average diameter. This value was reported for each replicate, where one value is the diameter of one bioprinted filament. GraphPad Prism v10.4.1 was used to chart this data, as well as to compare the diameter of the bioprint with and without microbubbles to determine if the microbubbles have an effect on filament diameter (unpaired t-test, N=3). A linear regression analysis was performed on all samples without microbubbles printed at 3 mm / s. [000181] Example 2: Coupling Ultrasound-Responsive Microbubbles to Plasmid DNA. [000182] The present disclosure demonstrates a method developed for coaxially bioprinting DNA-coupled ultrasound-responsive microbubbles in cell-laden bioinks, where focused ultrasound is applied to these bioprinted constructs resulting in spatiotemporally controllable gene delivery (Fig. 1). To characterize the microbubble gene delivery vehicles, cationic lipid coated microbubbles were fabricated and electrostatically coupled to plasmid DNA, and the loaded DNA was visualized using YOYO-1 nucleic acid stain. Microbubbles were imaged usingbrightfield (Figs. 2A and 2E) and fluorescence microscopy (Figs. 2B and 2F). By overlaying the brightfield and fluorescence images, we observed that the fluorescent signal of the YOYO-1- stained DNA localized to the microbubbles (Fig. 2C) and showed stable DNA retention even after repeated washing. Microbubble size distribution was quantified by image analysis of the brightfield images, showing that the population of the microbubbles were between 1-5 μm in diameter (Fig.2D). [000183] Example 3: Coaxial Bioprinting of Ultrasound-Responsive Microbubbles in Bioinks of Increasing Alginate Concentrations [000184] Microbubbles were added to varying concentrations of sodium alginate and coaxially bioprinted lattice constructs. Additionally, control constructs were printed without microbubbles for comparison. The 2% alginate (Fig.3A) required a printing pressure of 10-12kPa and was the most challenging bioink to print due to its low viscosity making it easy to over-extrude. The 4% alginate (Fig.3B) required a printing pressure of 11-13kPa and was the easiest to print and maintain print fidelity. Finally, the 6% alginate (Fig. 3C) required a printing pressure of 13-15kPa and was similar to the 4% alginate in print ease and fidelity. Bioprinted constructs were imaged using a digital camera, to capture the entire construct, as well as brightfield microscopy. [000185] The bioinks behaved similarly with and without microbubbles. At all alginate concentrations, the presence of the microbubbles did not cause any observable differences in printability or required extrusion pressure. The incorporated microbubbles are visible in the bioprinted filaments in the widefield macroscale views as the opaque white coloration. When viewed with trans-illumination bright field microscopy, the microbubbles scattered the light and caused the filaments to appear dark. When viewed at high magnification, individual microbubbles are visible as dark dots within the filaments. The filaments appear clear and without coloration when microbubbles are not present. As supported by the macroscale and microscopy filament images (Figs. 3A-3C), the microbubbles maintained stability throughout the printing and imaging process. [000186] Therefore, it was observed that higher concentrations of alginate used for microbubble-containing bioinks, such as 6% w / v, were more easily printable with the coaxial extrusion setup and maintained higher print fidelity than lower alginate concentrations, but even the 2% w / v alginate had readily achievable printability. These lower alginate concentrations were less viscous and required a lower extrusion printing pressure to maintain printability. [000187] The inclusion of ultrasound-responsive microbubbles was found to have no apparent effect on print fidelity, printability, or required printing pressure of alginate bioink formulations.The microbubbles maintained particle integrity in multiple alginate concentrations and were stable throughout printing and imaging. High printing pressures could jeopardize microbubble stability and cause them to prematurely collapse during the printing process, so it was ensured that the bioinks could be printed below 15kPa for even the highest concentration of alginate, which is desirable for both microbubble stability and cell viability.65[000188] Example 4: Stability of Ultrasound-Responsive Microbubbles in Bioprinted 4% w / v Alginate Constructs. [000189] After demonstrating the stable printing of the alginate bioinks with incorporated ultrasound-responsive microbubbles, it was determined how long the microbubbles remained stable in the printed constructs. Microbubbles were added to HEK293T-laden 4% w / v alginate, and filaments were coaxially bioprinted. The bioprints were placed either in the incubator at 37˚C, or at room temperature (RT). Immediately post-printing (0 hr), the bioprints were imaged to determine the starting pixel intensity of transmitted light through the filaments. Samples were then imaged at various timepoints over 48 hrs to determine how long the microbubbles remained stable (Figs.4A-4C). At 48 hrs, it was observed that over 70% of the microbubble scattering signal was still present. [000190] The study of microbubble stability in bioprinted cell-laden alginate constructs demonstrates the ability of ultrasound-responsive microbubbles to maintain particle integrity and responsiveness over time in the constructs after the printing process is concluded. Over 70% of the microbubble scattering signal was still present at 48 hr in both room temperature and 37°C conditions. The 37°C condition is valuable to support cell viability which enables user-defined temporal control over ultrasound-mediated gene delivery. This stability test was conducted at a microbubble concentration of 2.34x109μB / mL, and the results in Figures 8A-8C show that a 1.5- fold and 3-fold reduction in microbubble concentration can achieve transfection in the print. Thus, it is possible that microbubbles could be loaded at higher concentrations in anticipation of the reduction in particles over time so that the correct number of microbubbles would be present at the desired time of activation. [000191] Example 5: Ultrasound Activation of Microbubbles in 4% w / v Alginate Bioprinted Constructs. [000192] To investigate the effect that bioink microbubble concentration had on the ultrasound- induced region of microbubble activation, we varied the concentration of microbubbles in 4% w / v alginate (Figs.5A-5C) and imaged using brightfield microscopy before exposing eachfilament to 40 pulses of focused ultrasound. The prints were imaged again post-ultrasound exposure, and the size of the microbubble activation zone was assessed by image analysis (Fig. 5D). Overall, the filaments with the two lower microbubble concentrations, had larger activation zone sizes of 1587 ± 171 μm and 1496 ± 139 μm, respectively. The filaments with the highest microbubble concentration had a significantly smaller activation zone size of 873 ± 194 μm with incomplete ultrasound activation of the microbubbles in the focal zone. This supports an inverse trend between the microbubble concentration and the focal zone size; as the concentration of microbubbles increased, the size of the microbubble activation zone decreased. [000193] Example 6: Viability of Cells in Bioprints with Ultrasound-Responsive Microbubbles and Increasing Alginate Concentration. [000194] To ensure that cells with microbubbles can be bioprinted and maintain high cell viability at different alginate concentrations, HEK293T cells were printed in varying w / v sodium alginate containing microbubbles. Bioprinted constructs were stained 0 hr and 48 hr post printing. The cells in the bioprints maintained a high viability of over 85% in all alginate concentrations from 0 hr to 48 hr post-printing (Fig. 6). [000195] From the viability study, it was observed that the bioprinting process had minimal adverse effects on cell viability. In all bioprinted samples, regardless of alginate bioink concentration, cell viability was over 85% at both 0 hr and 48 hr post-printing. This indicates that the pressure required to print the samples as well as time spent out of the incubator did not significantly affect cell viability. Additionally, the constructs maintained high cell viability for over 48 hr post-printing, which is a sufficient timepoint to assess ultrasound-mediated cellular transfection. The bioprinting method minimizes the time necessary for the cell-laden bioinks to remain outside of the incubator and affords the ability to maintain a set temperature of 37°C during both the pre-printing and printing processes. Both the low printing pressures and minimized temperature variances could be factors attributing to the high viability of the disclosed bioprinted cells. [000196] Example 7: Effect of Varying the Number of Ultrasound Pulses on Ultrasound- Controlled Cellular Transfection in Coaxially-Bioprinted Constructs. [000197] The number of focused ultrasound pulses that a construct is exposed to is a parameter that affects microbubble activation, which in turn affects the number of transfected cells in the ultrasound focal zone. To investigate the ability to modulate transfection withultrasound exposure, bioprinted alginate filaments containing GFP plasmid microbubbles and HEK293T cells were exposed to varying numbers of focused ultrasound pulses and imaged post-ultrasound to visualize the region of microbubble activation. After 48 hours, the samples were fixed and stained with Hoechst nuclear stain (Fig. 7A). The number of transfected cells in each construct ranged from 2-30 cells per construct, with the fewest pulses (10) having an average of 2 transfected cells. The constructs exposed to 40 pulses had an average of 16 transfected cells, and the constructs exposed to 80 pulses had an average of 23 transfected cells (Fig. 7B). It was observed that as the number of ultrasound pulses increased, there was an increasing trend in the number of transfected cells within the bioprinted constructs. Upon measuring the diameter of the zone containing transfected cells along the filaments (the effective transfection zone diameter), there was no significant change observed in effective transfection zone diameter among the groups exposed to different numbers of ultrasound pulses (Fig. 7C). Control samples that were not exposed to ultrasound pulses did not show any evidence of transfection when imaged and analyzed at 48 hrs, indicating that ultrasound stimulus application is necessary for gene delivery (Fig. 10). In all ultrasound-exposed samples, the boundaries of the filaments remained visible and uniform in appearance at the 48 hr time point. [000198] Example 8: Effect of Varying Microbubble Concentration on Ultrasound- Controlled Cellular Transfection in Coaxially-Bioprinted Constructs. [000199] To determine the effect of microbubble concentration within the cell-laden bioprints on the number of ultrasound transfected cells and the size of the zone in which cells are transfected, GFP-plasmid loaded microbubbles were added at multiple concentrations to 4% alginate containing HEK293T cells and coaxially bioprinted filaments as previously described. After 48 hours, the samples were analyzed for GFP expression by fixing and staining with Hoechst nuclear stain and then imaged with fluorescence and brightfield microscopy (Fig. 8A). [000200] Microbubble concentration was observed to affect the number of transfected cells, with the highest average transfection occurring at the intermediate concentration of 1.56x109μB / mL. Transfection was observed to decrease as microbubble concentration increased above this (Fig. 8B). The lowest microbubble concentration was observed to have the largest diameter zone of transfected cells (Fig. 13), and increasing microbubble concentration showed a decrease in the diameter of the transfection zone (Fig. 8C). Control samples containing no microbubbles that were exposed to ultrasound showed no transfected cells. This indicates the presence of gene delivery microbubbles is necessary for DNA delivery (Fig. 11). Additionalcontrol samples containing microbubbles with no GFP-plasmid were treated with ultrasound exposure and no transfected cells were observed (Fig.12). [000201] Thus, microbubble concentration was found to inversely affect the size of the ultrasound activation zone of the microbubbles with lower concentrations of microbubbles having larger regions of activation. It was also observed that microbubble concentration affects the number of transfected cells and the diameter of the transfection zone. The lowest concentration of microbubbles had the highest diameter region where the cells were transfected following focused ultrasound exposure. It has been reported that at high microbubble concentrations, the density of microbubbles can create a shielding effect reflecting some of the ultrasound energy thereby shielding microbubbles from the full ultrasound intensity. Microbubbles closest to the transducer are the least shielded and collapse first.13As these microbubbles disappear, there is less of a shielding effect each time the construct is exposed to ultrasound.61This shielding effect explains the consistent observation that higher concentrations of microbubbles led to fewer microbubbles being activated via focused ultrasound, and therefore smaller microbubble activation zones compared to constructs with lower microbubble concentrations. Microbubble collapse dynamics can also be influenced by the distance between adjacent microbubbles48which changes with increasing concentrations loaded in the filament and could play a role in the activation differences observed. [000202] Also, the number of transfected cells had an increasing trend as the microbubble concentration increased to 1.56 x 109μB / mL, but transfection decreased as microbubble concentration was further increased. A potential explanation for this is that at lower microbubble concentrations, there was less shielding and all of the ultrasound-responsive microbubbles in the construct were activated. As microbubble concentration started to increase, more bubbles were activated increasing the chances of successful cell transfection. As microbubble concentration increased further, it reached a point where the shielding effect actually reduced the number of activated microbubbles, thus reducing cellular transfection. This is consistent with the brightfield images showing that post-ultrasound, no microbubbles are seen to remain in the construct with the lowest starting microbubble concentration, but more microbubbles are present at the higher concentrations. [000203] Example 9: Modulation of Osteoblast Transfection in Coaxially-Bioprinted Constructs. [000204] Extending the technique of focused ultrasound-mediated genetic delivery in bioprints to additional cell types, osteoblasts (hFOB 1.19) were incorporated into the ultrasound-responsive bioinks and the number of ultrasound pulses applied were varied (Fig. 9A). It was observed that as the number of ultrasound pulses increased, there was an increasing trend in the number of transfected cells. The number of transfected cells in each construct ranged from 12-32 cells per construct, with the fewest pulses (10) having an average of 14 transfected cells (Fig. 9B). The constructs exposed to 40 pulses had an average of 18 transfected cells, and the constructs exposed to 80 pulses had an average of 31 transfected cells. There was no significant change observed in effective transfection zone diameter among the groups exposed to different numbers of ultrasound pulses (Fig. 9C). Control samples that were not exposed to ultrasound pulses did not show any evidence of transfection when imaged and analyzed at 48 hrs, indicating that ultrasound stimulus application is necessary for gene delivery (Fig. 14). Osteoblasts printed with no microbubbles and no plasmid, also showed no visible transfection with applied ultrasound pulses (Fig.15). [000205] Sonoporation is a physical process requiring no chemicals or active cellular processes, so it is a promising technique for genetic manipulation that can be applied to a wide range of cell types.34,57It was observed in both bioprinted HEK293T cells and bioprinted osteoblasts (hFOB 1.19) that as the number of focused ultrasound pulses applied to a bioprinted filament increased, the number of transfected cells increased, and the diameter of the zone of cell transfection increased. As more pulses of ultrasound are applied, fewer microbubbles remain in the sample to aid the shielding effect, which results in the activation of more microbubbles with each additional ultrasound pulse. This demonstrates that the number of transfected cells can be modulated by the number of focused ultrasound pulses applied to the bioprinted construct. The printed constructs containing osteoblasts had consistently higher numbers of transfected cells compared to the HEK293T cells (shown in Example 7), regardless of ultrasound pulse quantity. A potential explanation for this is that as the surface area of the cell increases, more microbubbles can interact with the cell surface, causing more transfection. Osteoblasts have a mean diameter of 25 μm50, and HEK293T cells are smaller with a mean diameter of 15 μm49. It was observed that the diameter of the region containing transfected cells also had an increasing trend with the number of ultrasound pulses where a ~2.5x increase in diameter was observed from 10 to 80 pulses. It is worth noting that these transfection results are consistent with the observations that as microbubble concentration increased the visually observed region of microbubble activation decreased, which is also likely due to the microbubble shielding effect. The results showed robust control over the number of transfected cells in a bioprint, from about 2 to 30 cells, which demonstrates the ability to modulate thenumber of transformed cells, important for studying diseases like cancer that initiate from transformations in a single cell or small cluster of cells.11,35,45[000206] Example 10: Transfection of multicellular structures in Bioprinted Constructs. [000207] The materials and methods for fabricating microbubbles, coupling of plasmid-DNA to the microbubbles, and preparation of the 4% alginate are the same as for HEK293T single cells as described previously in Example 1. [000208] Preparing LifeSupport® for FRESH Printing: MCF10A (non-tumorigenic breast epithelial cell) media is prepared and calcium chloride (CaCl2) is added for a final concentration of 0.2% CaCl2and mixed well by vortexing.500 mg of LifeSupport® powder is placed into a 50 mL centrifuge tube and 10mL of 4°C MCF10A media+0.2% CaCl2is added and vortexed well for 1 minute. The LifeSupport® slurry is placed at 4°C for 15 minutes, then centrifuged at 2100RCF for 7 minutes at 21°C. ~300μL of LifeSupport® is then added to the corner well of an 8-well LabTek 1.0 coverglass bottom dish. [000209] Fabricating MCF10A spheroids: Breast epithelial cells (MCF10A) were added to 96- well Nunclon low-adhesion u-bottom plates at a concentration of 3000 cells / well. The plates were centrifuged at room temperature, 400RPM, for 5 minutes, then again for 3 minutes. This additional spin helps to produce consistently round spheroids. The cells are cultured in the incubator for 4 days before usage. [000210] Coupling plasmid-loaded microbubbles to spheroids: The spheroids are removed from the u-bottom plate via pipetting and placed into a 50mL centrifuge tube. Four plates of spheroids are used for each 400μL of complete (alginate / microbubble / spheroid) bioink. After all the spheroids are in the centrifuge tube, the tube is placed into the incubator for 10 minutes to allow all spheroids to sink to the bottom. The additional media is then carefully removed via vacuum with a glass pipette, being careful not to aspirate the spheroids. Plasmid-loaded microbubbles are diluted from their stock concentration to an equal final concentration of 7.56x109μB / mL in the 400uL of complete bioink. The microbubbles in -Ca / -Mg DPBS are added to the tube containing spheroids and gently hand-flicked to mix every 2 minutes for 10 minutes. This pre-coupling ensures that microbubbles are in close proximity to the spheroids. [000211] Incorporating microbubble-coupled spheroids into alginate bioinks: Alginate bioinks were prepared as previously stated, and 400μL of the alginate was added to a 3mL bioprinting syringe (CellInk). The previously-coupled spheroid / microbubbles are then added to the alginate for a final concentration of 7.56x109μB / mL with 384 spheroids (4 x 96-well plates ofspheroids) and are stirred gently to avoid the incorporation of air bubbles using a 22G blunt-tip needle attached to a syringe. [000212] FRESH Bioprinting cell-laden ultrasound-responsive alginate bioinks: The CellInk Bio-X6 bioprinter with DNA Studio 4.0 software was used throughout all experiments. The previously prepared syringe containing the cell-laden ultrasound-responsive alginate bioink was attached to a conical 22G printing tip and placed at 37˚C in the bioprinter. The bioprinter was calibrated to touch the surface of the coverglass in the LabTek 8-well dish and then moved upward 0.2mm to give clearance space for the print to extrude. The bioink is printed at 4mm / s at 37˚C (for cell viability) in the range of 7-11 kPa. A pre-flow delay of -400ms and a post-flow delay of -400ms was used to aid print fidelity. Both of the flow delays are negative values, meaning that the pre-flow delay causes the ink to extrude before the nozzle begins moving, and the post-flow delay causes the ink to stop extruding before the nozzle stops. [000213] Removal of LifeSupport®: After printing into the support bath, the prints are partially crosslinked due to the added calcium chloride in the LifeSupport®. An additional 200μL of 37°C MCF10A media+0.2% CaCl2is added to the well to promote additional crosslinking. The dish is placed at 37°C for 10 minutes to dissolve in the support bath, which is then carefully removed from the dish via pipetting. 400μL of MCF10A growth media containing no added calcium chloride is added to the dish and placed back into the incubator for 5 minutes to dissolve any remaining LifeSupport®. This media is then carefully removed from the dish via pipetting. [000214] The materials and methods for casting the bioprints in 2.7mg / mL collagen type 1, and exposure to 80 pulses of focused ultrasound is the same as for HEK293T single cells as described previously in Example 1. [000215] Results: Multicellular breast epithelial cell (MCF10A) spheroids were incorporated with GFP-plasmid loaded microbubbles into an alginate bioink as depicted in Fig. 20. Support bath-mediated FRESH (Freeform Reversible Embedding of Suspended Hydrogels) bioprinting was used to print a lattice construct containing the microbubbles and spheroids (Fig. 19E). Upon exposure to 80 pulses of focused ultrasound in the center of the construct, microbubbles were seen to rupture and activate in the ultrasound-exposed region (Fig. 19F). At 48hrs after ultrasound exposure, spheroid cells from within the ultrasound-exposed area were seen to be transfected with GFP plasmid (Figs. 19G, 19H). [000216] In a separate experiment, MCF10A breast epithelial spheroids were incorporated into alginate bioinks with microbubbles loaded with a plasmid coding for fluorescently tagged HER2 oncogenic protein and printed into lattice structures using the FRESH technique (Fig. 19I). Microbubbles were again seen to rupture and activate in response to focused ultrasoundapplied in the center of the construct (Fig. 19J). At 48hrs after ultrasound exposure, spheroid cells from within the ultrasound-exposed area were seen to be transfected with the HER2 plasmid (Fig.19K), where HER2 shows characteristic expression on the cell surface. The ability to transfect individual breast epithelial spheroid cells with the HER2 oncogene can allow for modeling and studying cancer progression from the first few cells within a healthy tissue that express the cancer protein. [000217] Example 11: Freeform Bioprinting of Ultrasound-Responsive Bioinks for Programmable Gene Delivery. [000218] Herein, a new technique is described to FRESH bioprint spatially complex ultrasound- responsive tissue constructs and utilize focused ultrasound to deliver genes to specified regions of the constructs with spatiotemporal control. Ultrasound-responsive microbubbles are electrostatically coupled with plasmid DNA and incorporated into a sodium alginate bioink along with individual cells or spheroids (Fig. 21A). This bioink is extrusion bioprinted into a support bath of gelatin beads doped with calcium chloride using a layer-by-layer approach (Fig. 21B). Sodium alginate crosslinks upon contact with calcium chloride, which allows the multilayer construct to remain freestanding but stationary for enhanced print fidelity. After printing, the dish is placed at 37°C to dissolve the support bath, leaving behind the printed tissue construct that can be further cultured. Upon ultrasound application, the microbubbles within the focal zone are activated, releasing coupled DNA to cells within the focal zone. This results in a user-defined region of genetic manipulation that can be spatially and temporally controlled (Fig.21C). The approach enables controlled genetic manipulation of specific regions of complex bioprinted structures. A first example of ultrasound-mediated targeted gene delivery in hollow bioprinted constructs has been demonstrated here, which allows genetic manipulation of architectures relevant to vasculature and tissue ducts. Using this technology, the ability to localize delivery of different genes in distinct user-defined regions of the print has been further demonstrated, enabling ultrasound-controlled expression of multiple transgenes. This method can be extended to spheroid models, where bioprinted spheroids in microbubble-laden prints are exposed to focused ultrasound. This results in a small subset of cells within the spheroid expressing a gene of interest such as oncogene, allowing modeling initiation of disease processes in bioprinted tissue microenvironments (Fig.21D). This technology provides a platform for controlling gene expression in engineered tissues to study genetic drivers of disease and instruct cell behavior. This system can become a uniquely capable tool for studying tumorigenesis and genetic driversof disease in 3D model systems, as well as coordinating functional cell processes for regenerative medicine applications. [000219] Example 12: Characterization and FRESH Bioprinting of Ultrasound-Responsive Alginate Bioinks. [000220] To bioprint ultrasound-responsive tissue constructs, the printing method must minimize printing pressure to ensure microbubble stability and allow for the extrusion of bioinks into volumetric multilayer constructs with minimal interface separation between printed layers. FRESH bioprinting allows for extrusion printing of multi-layer tissue constructs into a support bath that immediately crosslinks the alginate bioink to form a single construct. The removal of the support bath at 37°C is well-suited for cellular viability and maintaining microbubble stability, and the low pressures required for FRESH printing are amenable to high microbubble stability while maintaining print fidelity. To assess sodium alginate bioink printability and fidelity, FRESH bioprinting was used to bioprint a 3x3 lattice model. As the alginate bioink is extruded and contacts the support bath containing 0.2% CaCl2, the printed filaments immediately begin to crosslink. However, the print is completed quickly enough to allow filaments contacting each other to partially crosslink together. Upon printing completion, the bioprinted lattice in the support bath is transferred to 37°C to dissolve the support bath, leaving a 3D tissue construct that can be cultured (Fig. 22A). Bioprinting ultrasound-responsive microbubbles requires low shear stress to maintain microbubble stability and maintain cellular viability. This shear stress can be controlled by varying the density of the bioink; however, low-viscosity inks can be difficult to bioprint with high fidelity. Additionally, the effects of microbubble presence on FRESH-printed alginate printability have not been previously characterized. Initial printing was performed of alginate lattices at varying concentrations, with and without microbubbles at 2.34 x 109μB / mL. Ease of printability increased with increasing alginate concentration (2% w / v, 4% w / v, and 6% w / v alginate), and microbubbles remained stable in all concentrations throughout the printing process and did not affect bioprinting fidelity. Higher alginate concentrations are less fluid before crosslinking, mitigating issues of premature extrusion and excess extrusion (Fig. 22B). Microbubbles remained intact through the 48 hr post-printing timepoint. Rheological characterization of alginate bioinks showed that all concentrations of alginate exhibited shear- thinning properties with or without microbubble inclusion. Presence of microbubbles did not alter the shear-thinning behavior of the bioinks, which is important for bioprinting 3D structures with high cellular viability (Fig. 22C).[000221] Ultrasound-mediated gene delivery is enabled by electrostatically coupling microbubbles with plasmid DNA carrying the transgene of interest. The plasmid-coupled microbubbles were stained with YOYO-1 nucleic acid stain to visualize the surface-loaded DNA. Microbubbles were imaged using brightfield and fluorescence microscopy, and the DNA- associated fluorescence was seen to localize with the outer surface of the microbubbles, maintaining electrostatic DNA retention after repeated washes (Figs. 22D and 22E). These results demonstrated the capability of FRESH bioprinting microbubble-containing alginate bioinks at multiple alginate concentrations, while maintaining microbubble stability and the mild shear-thinning characteristics expected for alginate, and allowing dispersion of the microbubbles throughout the print. [000222] Example 13: Localized Gene Delivery in FRESH-Bioprinted Ultrasound- Responsive Constructs. [000223] After having established the ability to print microbubbles using the reversible support- bath mediated method, next the ultrasound-responsivity of the bioprinted constructs was assessed. A 3x3 lattice structure comprising alginate with ultrasound-responsive microbubbles was bioprinted. Once the printed construct had been released from the support bath slurry, it was cast in 2.7 mg / mL neutralized type I collagen. Casting the construct in collagen is important for facilitating ultrasound-mediated gene delivery, as it both holds the print in place and minimizes interface differences that can scatter the focused ultrasound beam. After crosslinking the collagen, the print was then exposed to a focused ultrasound beam that selectively activates the microbubbles in the focal region (Fig. 23A). To assess the region of microbubbles activated via ultrasound, alginate filaments were bioprinted with increasing alginate concentration (2% w / v, 4% w / v, and 6% w / v) containing microbubbles at 2.34 x 109μB / mL and the center region of the filament was exposed to focused ultrasound (Fig. 23B). As the concentration of alginate increased from 2% to 6% w / v, the size of the microbubble activation region increased from an average of 1298 ± 38μm to 1642 ± 32μm (Fig. 23C). This may suggest that the 2% and 4% w / v alginate provide better impedance matching to the collagen hydrogel which could contribute to tighter spatial localization of the ultrasound and less dispersal of the focused beam. This data supported utilizing 4% w / v sodium alginate for the bioink of choice due to its printability, rheological properties, and tight ultrasound activation region. [000224] Following printing of an ultrasound-responsive bioink composed of 4% w / v sodium alginate, cells, and DNA-coupled microbubbles into a lattice construct; focused ultrasound can be applied to a user-defined region to selectively activate microbubble gene delivery vehicles.Upon ultrasound exposure, microbubbles release their genetic cargo and local cells undergo sonoporation which facilitates the genetic payload into the cells (Fig.23D). To demonstrate this principle, filaments of alginate bioink were bioprinted containing microbubbles coupled with a GFP plasmid and HEK293T cells and they were exposed to varying pulses of ultrasound. Transfected cells ranged from 15 ± 5 cells with 10 ultrasound pulses to 117 ± 55 cells with 80 ultrasound pulses, showing the ability to control the number of transfected cells with ultrasound pulses applied. A significant increase was seen between the lowest and highest number of pulses applied, which was correlated with a larger visible region of ultrasound activation. The ability to modulate the number of transfected cells is valuable to control the degree of genetic manipulation a tissue construct undergoes. Focused ultrasound allows the user to define both the spatial location and magnitude of gene delivery, with the amount of applied ultrasound controlling the magnitude of the manipulation. [000225] To further illustrate, a lattice structure from the same bioink was bioprinted. The printed construct was imaged immediately post-printing, after ultrasound application, and after nuclear staining done at 48hr post-printing. The region of microbubble activation was localized to the center of the ultrasound focal zone, as was the genetic manipulation shown by cells expressing GFP. This region of gene delivery was highly focal within the larger print (Fig.23E) and demonstrated the ability to activate transfection at filament junctions. Additional control experiments showed there was no transfection present in samples without microbubbles, without ultrasound, or with vehicle control particles only. Both the presence of plasmid-coupled microbubbles and ultrasound stimulation are necessary to facilitate localized transfection in 3D bioprinted constructs, which allows for highly spatially-defined and user-defined gene delivery. [000226] Example 14: Ultrasound-Patterned Gene Delivery in Multi-Ink and Tubular Bioprinted Architectures. [000227] Utilizing multiple bioinks containing microbubbles with different genetic payloads can allow for on-demand and site-specific control of ultrasound-mediated gene delivery of multiple transgenes. By bioprinting filaments containing microbubbles coupled with a genetic payload to cause GFP expression (“GFP microbubbles”), in alternation with filaments containing microbubbles coupled with another genetic payload for mCherry expression (“mCherry microbubbles”), this multiplexing was illustrated. Once this construct was released from the support bath and cast in neutralized collagen, the focused ultrasound was rastered across the printed construct in a defined pattern (Fig.24A). This resulted in patternable gene delivery, with GFP cells and mCherry cells localized in their respective filaments. By bioprinting 4% w / valginate filaments containing HEK293T cells and either GFP microbubbles or mCherry microbubbles in alternation and exposing to focused ultrasound, defined regions of microbubble activation were seen and gene patterning was observed that are highly localized (Fig. 24B). Creating complex structures that can more accurately represent the tissue’s native environment is useful for accurately modeling disease states. The ability to create hollow tubular structures enables fabricating architectures relevant to vasculature or other perfusable networks, or tissue ducts. Tubular vessel-like structures were bioprinted using the support-bath mediated technique with 4% w / v alginate and GFP plasmid-loaded microbubbles embedded within the tube walls (Fig. 24C). After focused ultrasound is applied to a region of the bioprinted construct, cells within that region begin overexpressing GFP localized to the ultrasound focal zone (Fig.24D). The localization is further highlighted in the size view of the tube, where the ultrasound was targeted to the top portion of the printed tube. This is the first demonstrated example of ultrasound- mediated gene delivery in a hollow bioprinted construct. Furthering this technique, a bifurcated tubular structure was bioprinted to showcase the ability to deliver genes to distinct regions of the printed construct with minimal off-target effects (Figs. 24E-24H). With focused ultrasound application, the ability to activate transfection in a particular region of the heterogenous bifurcated structure was shown (Fig. 24I). The ability to bioprint multi-ink volumetric tissue constructs is useful for more accurately representing the microenvironment of native tissue. Different cell types can be incorporated into the bioinks, and microbubbles with different genetic payloads can be used to transform tissue constructs with spatial and temporal control. The ability to genetically manipulate cells in different regions of the same tissue microenvironment has applications in tissue regeneration and wound healing. For example, this technique could be used to locally induce secretion of growth factors from vascular cells to promote vascularization, while in another region of the same bioprinted construct, differentiation factors can be delivered to stem cells, with the additional ability for on-demand control of the delivery. [000228] Example 15: Ultrasound-Mediated Gene Delivery in FRESH-Bioprinted Breast Epithelial Spheroids for HER2 Overexpression. [000229] The use of multicellular spheroids for bioprinting is valuable for modeling intracellular connections to create more relevant tissue models. To bioprint spheroids, the same support- bath mediated procedure was followed as previously described, but with the plasmid-loaded microbubbles pre-coupled to the spheroids to ensure close proximity. The spheroids coupled with microbubbles are then added to the alginate ink, where they are bioprinted and processed as previously described. Focused ultrasound is applied to the bioprinted spheroid structure, withthe aim to transfect a select number of cells within spheroids in the ultrasound focal zone to express the gene coupled to the microbubbles (Fig. 25A). This technique is relevant for modeling initiation of diseases such as cancer that begin from a single or small subset of cells. Using microbubbles coupled with a plasmid for GFP expression, the number of transfected cells within a spheroid can be modulated by varying the number of applied ultrasound pulses (Fig. 25B), and increasing the number of pulses from 10 to 80 increases the number of transfected cells from an average of 1 to 9 transfected cells per spheroid (Fig. 25C). The ability to modulate the number of transfected cells is useful for modeling processes such as tumorigenesis where the initiation occurs within mature tissue that has established intracellular signaling and connections and can begin with genetic and epigenetic changes in a single cell or subset of cells. Bioprinted spheroids maintained high viability for up to 4 days post-printing, and the application of focused ultrasound did not affect viability. [000230] To further investigate the capabilities of this technique, spheroids with microbubbles coupled with a plasmid to induce HER2 overexpression were bioprinted and were exposed to focused ultrasound. Upon ultrasound stimulation, between 1-4 HER2 overexpressing cells per spheroid were observed and a characteristic membrane expression of the HER2 protein was seen in all transfected cells (Fig. 25D). HER2 overexpressing cells forming protrusions to interact with their microenvironment was also observed, which has been previously found in cancer patient tissue sections (Fig. 25D). [000231] Example 16: Ultrasound-mediated gene delivery in FRESH bioprinted constructs with smaller-diameter filaments. [000232] The bioprinting parameters of pressure and speed for alginate bioinks without embedded microbubbles with varying needle gauges were first assessed. FITC-dextran was added to the alginate bioink to enhance visibility for analysis and did not affect the shear thinning properties of the bioink. For each needle gauge, 4% w / v alginate was bioprinted with varying pressures and speeds to determine which combination of pressure and speed produced a bioprinted filament diameter closest to the theoretical value of the needle gauge's inner diameter (Table 1). In general, the bioprinted filaments resemble the theoretical values expected from the inner diameters of the printing needles. With smaller gauge needles, the overall diameter was smaller than the theoretical values; however, this itself can be useful as there is a need to bioprint feature sizes less than 200 μm to mitigate issues of nutrient diffusion in larger constructs. Alginate bioink containing ultrasound-responsive microbubbles was then bioprinted at the established pressure and speed conditions. Interestingly, the presence of microbubblesgenerally resulted in the average filament diameter more closely matching the theoretical values, except for the largest 20G needle. Table 1. Optimized bioprinting parameters for bioinks with and without microbubbles using printing needle gauges ranging from 20 – 30G., affects print fidelity; the widths of the filaments were assessed at pressures 2 kPa above and below the determined optimal pressure. For larger diameter needle gauges, such as 20G and 22G, as pressure increased filament diameter increased. However, the smaller needle gauges (25G, 27G, and 30G) had similar filament diameters with higher pressures. For all needle gauges at varying pressures, there were no significant changes in filament diameter when microbubbles were embedded in alginate bioinks. This is advantageous, as lower bioprinting pressures result in lower shear stress exerted on cells and is more amenable to cell viability. [000234] To assess how bioprinting speed parameter affects the bioprinted filament diameter, the alginate bioink was printed at the previously determined optimal printing pressure with speeds of 1 mm / s above and below the determined optimal speed. Generally, as speed increased the bioprinted filament diameter decreased. This is due to the printhead being moving too quickly for the rate of extrusion and would require higher pressures to extrude the bioink rapidly enough to achieve target filament diameters. However, in smaller gauge needles (27, 30G), bioprinting speed did not have a significant impact on the filament diameter. In the speed parameter assessment, there was no significant change in filament diameters for all gauges of print needles when microbubbles were incorporated in the alginate bioink. [000235] Upon determination of optimal bioprinting parameters for microbubble-laden alginate bioinks, the optimal bioprinting conditions were used for the median gauge needle (25G) incorporated with HEK293T cells as previously described. The bioprinted cells maintained high viability (94% ± 1%) for 48 hours post printing. The microbubbles were coupled with plasmid DNA to cause expression of GFP in transfected cells. The bioink was printed into a support bathcontaining calcium chloride to facilitate rapid crosslinking of the alginate. Upon bioprinting completion, the support bath was dissolved at 37°C and the filament was cast in 2.7mg / mL neutralized type I collagen. This facilitates ultrasound-mediated gene delivery by immobilizing the bioprinted filament and reducing ultrasound scattering by minimizing interface differences. The center of the construct was then exposed to 10 pulses of focused ultrasound to activate the microbubbles within the focal zone, resulting in a region of transfected cells within the ultrasound focal zone (Fig. 26A). After 48 hours, a patterned region of ultrasound-mediated gene delivery was visible in the ultrasound focal zone, with an average of 30 ± 12 (N=3) transfected GFP-expressing cells per bioprint. The bioprinted construct was imaged before, immediately after, and 48 hours post-ultrasound exposure. Hoechst nuclear stain was used to visualize bioprinted cells (Fig. 26B). (VI) References 1. Alemany-Ribes, M., and C.E. Semino. Bioengineering 3D environments for cancer models. Adv. Drug Deliv. Rev. Netherlands, 79–80:40–49, 2014. 2. Ash, C., M. Dubec, K. Donne, and T. Bashford. 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Mechanisms underlying sonoporation: Interaction between microbubbles and cells. Ultrason. Sonochem. 67:105096, 2020. 68. Zhang, Z.-Z. et al. Role of scaffold mean pore size in meniscus regeneration. Acta Biomater. 43:314–326, 2016. 69. Zhao, Z., and P.W. Fairchild. Dependence of light transmission through human skin on incident beam diameter at different wavelengths. In: Laser-Tissue Interaction IX, edited by S.L. Jacques. pp.354–360, 1998. 70. Zhi, D. et al. A review on cationic lipids with different linkers for gene delivery. Adv. Colloid Interface Sci.253:117–140, 2018. [000236] Each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” The transition term “comprise” or “comprises” means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. [000237] Unless otherwise indicated, all numbers expressing quantities of ingredients, agent concentrations, experimental conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value. [000238] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. [000239] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention. [000240] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims. [000241] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. [000242] Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching. [000243] It is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described. [000244] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. [000245] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the example(s) or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster'sDictionary, 11th Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology, 2nd Edition (Ed. Anthony Smith, Oxford University Press, Oxford, 2006).

Claims

CLAIMS 1. A composition comprising: a hydrogel scaffold with a plurality of filament structures printed on a substrate; and a collagen cast gel that encases the plurality of filament structures of the hydrogel scaffold; wherein the composition is configured to receive a population of microparticle-coupled cargo for controlled delivery to one or more target cells by application of ultrasound energy.

2. The composition of claim 1, wherein the filament structures are printed adjacent to each other and / or on top of one another such that the filament structures form a cross-bridge.

3. The composition of claim 1, wherein the filament structures are printed in any suitable geometry selected from the group of circles, squares, rectangles, triangles, polygons, laminar geometries, and irregular geometries, or any combinations thereof.

4. The composition of any of claims 1-3, wherein the hydrogel scaffold comprises an agent selected from the group of alginate, fibrin, agarose, chitosan, gellan gum, cellulose, hyaluronic acid, gelatin methacryloyl, and methacrylated collagen, or any combinations thereof.

5. The composition of claim 1, wherein the collagen cast gel is bioprinted.

6. The composition of any of claims 1-5, wherein at least one of the filament structures from the plurality of filament structures of the hydrogel scaffold further comprises the population of microparticle-coupled cargo.

7. The composition of claim 6, wherein the population of microparticle-coupled cargo is distributed over an area of at least one of the filament structures of the hydrogel scaffold.

8. The composition of any of claims 1-7, wherein the population of microparticle-coupled cargo comprises a population of gas-filled microbubbles coupled to a substance selected from the group of nucleic acid, peptide, polypeptide, amino acid, protein, and drug, or any combinations thereof.

9. The composition of claim 8, wherein the substance is coupled to the gas-filled microbubbles via an electrostatic interaction, covalent bond, hydrogen bond, van der Waals force, or conjugation.

10. The composition of any of claims 1-9, wherein the one or more target cells comprise mammalian single cells or cell aggregates dispersed in at least one of the filament structures of the plurality of filament structures of the hydrogel scaffold.

11. The composition of any of claims 1-10, wherein the population of microparticle-coupled cargo and the one or more target cells present in the hydrogel scaffold are in close proximity to each other.

12. The composition of any of claims 1-11, wherein the application of ultrasound energy causes cavitation of the microparticles at a focused region of the hydrogel scaffold to deliver the cargo to the one or more target cells in proximity to the cavitated microparticles within the hydrogel scaffold.

13. A composition comprising: a hydrogel scaffold with a plurality of filament structures; a collagen cast gel encasing the plurality of filament structures of the hydrogel scaffold; a population of mammalian cells incorporated into the hydrogel scaffold; and a plurality of microbubbles dispersed throughout the filament structures of the hydrogel scaffold, wherein the microbubbles are attached to one or more nucleic acid molecules to be expressed in the mammalian cells.

14. The composition of claim 13, wherein the hydrogel scaffold comprises an agent selected from the group of alginate, fibrin, agarose, chitosan, gellan gum, cellulose, hyaluronic acid, gelatin methacryloyl, and methacrylated collagen, or any combinations thereof.

15. The composition of claim 14, wherein the hydrogel scaffold comprises alginate.

16. The composition of any of claims 13-15, wherein the collagen cast gel is selected from the group of type I collagen, type II collagen, and type III collagen, or any combinations thereof.

17. The composition of claim 16, wherein the collagen cast gel comprises type I collagen.

18. The composition of any of claims 13-17, wherein the plurality of microbubbles and the population of mammalian cells incorporated into the hydrogel scaffold are in close proximity to each other.

19. The composition of any of claims 13-17, wherein the plurality of microbubbles dispersed throughout the filament structures of the hydrogel scaffold is electrostatically attached to a surface of the mammalian cells incorporated into the hydrogel scaffold.

20. The composition of any of claims 13-19, wherein each microbubble of the plurality of microbubbles comprises an envelope and a gas core.

21. The composition of claim 20, wherein the envelope of each microbubble comprises a lipid monolayer or multilayer.

22. The composition of claim 20, wherein the gas core of each microbubble comprises a gas selected from the group of perfluorocarbon, carbon dioxide, nitrogen, nitrous oxide, helium, argon, nitric oxide, xenon, carbon monoxide, oxygen, and isoflurane, or any combinations thereof.

23. The composition of claim 22, wherein the gas core of each microbubble comprises perfluorocarbon gas.

24. The composition of any of claims 13-23, wherein each microbubble of the plurality of microbubbles has a diameter ranging from about 0.05 μm to about 5 μm.

25. The composition of claim 24, wherein each microbubble of the plurality of microbubbles has a diameter ranging from about 1 μm to about 5 μm.

26. The composition of any of claims 13-25, wherein the one or more nucleic acid molecules attached to the microbubbles are selected from the group of a linear DNA, circular DNA, cDNA, miRNA, shRNA, siRNA, plasmid, viral construct, and transposase construct, or any combinations thereof.

27. The composition of any of claims 13-26, wherein the one or more nucleic acid molecules are attached to the microbubbles via electrostatic interaction.

28. The composition of any of claims 13-27, wherein the population of mammalian cells comprises single cells scattered throughout the filament structures of the hydrogel scaffold.

29. The composition of claim 28, wherein the single cells of the population of mammalian cells comprise HEK293T cells.

30. The composition of claim 28, wherein the single cells of the population of mammalian cells comprise osteoblasts.

31. The composition of any of claims 13-27, wherein the population of mammalian cells comprises cell aggregates incorporated throughout the filament structures of the hydrogel scaffold.

32. The composition of claim 31, wherein the cell aggregates comprise spheroids or organoids.

33. The composition of claim 32, wherein the spheroids comprise breast epithelial spheroids.

34. The composition of any of claims 13-33, wherein the composition is configured to be responsive to an ultrasonic frequency ranging from about 0.5 MHz to about 15 MHz.

35. The composition of claim 34, wherein the ultrasonic frequency causes the microbubbles to implode thereby delivering the one or more nucleic acid molecules to the mammalian cells for expression.

36. The composition of claim 35, wherein the delivery of the one or more nucleic acid molecules to the mammalian cells occurs at a localized region of the hydrogel scaffold.

37. A composition comprising:a hydrogel scaffold with a plurality of filament structures; a collagen cast gel encasing the plurality of filament structures of the hydrogel scaffold; a population of mammalian cells incorporated into the hydrogel scaffold; a plurality of microbubbles dispersed throughout the filament structures of the hydrogel scaffold; and a plurality of nucleic acid molecules distributed over an area of at least one of the filament structures of the hydrogel scaffold to be expressed in the mammalian cells.

38. The composition of claim 37, wherein the hydrogel scaffold comprises an agent selected from the group of alginate, fibrin, agarose, chitosan, gellan gum, cellulose, hyaluronic acid, gelatin methacryloyl, and methacrylated collagen, or any combinations thereof.

39. The composition of claim 37 or claim 38, wherein the mammalian cells, the plurality of microbubbles and the plurality of nucleic acid molecules are in close proximity to each other within the filament structures of the hydrogel scaffold.

40. The composition of any of claims 37-39, wherein the mammalian cells, the microbubbles and the nucleic acid molecules present within the filament structures of the hydrogel scaffold are devoid of any interactions.

41. The composition of any of claims 37-39, wherein the microbubbles are attached to the nucleic acid molecules via an electrostatic interaction to form a microbubble-coupled cargo.

42. The composition of claim 41, wherein the microbubble-coupled cargo lacks interaction with the mammalian cells within the hydrogel scaffold.

43. The composition of claim 41, wherein the microbubble-coupled cargo is further attached to a surface of the mammalian cells within the hydrogel scaffold via electrostatic interaction.

44. The composition of any of claims 37-43, wherein a distance between the mammalian cells and the microbubbles within the hydrogel scaffold ranges from about 0 μm to about 35 μm.

45. The composition of any of claims 37-44, wherein the population of mammalian cells comprises single cells or cell aggregates incorporated into the filament structures of the hydrogel scaffold.

46. The composition of any of claims 37-45, wherein the composition is configured to be responsive to an ultrasound frequency ranging from about 0.5 MHz to about 15 MHz.

47. The composition of claim 46, wherein the ultrasound frequency causes cavitation of the microbubbles at a focal zone to deliver the nucleic acid molecules to the mammalian cells in proximity to the cavitated microbubbles for expression within the hydrogel scaffold.

48. The composition of claim 47, wherein a size of the focal zone of the hydrogel scaffold ranges from about 0.3 mm to about 2 mm.

49. A method of creating a transgenic target cell, the method comprising the steps of: (a) providing a hydrogel scaffold with a plurality of filament structures encased in a collagen cast gel, wherein the filament structures comprise a plurality of target cells and a population of ultrasound-responsive microbubbles stably bound to one or more nucleic acid molecules; (b) applying ultrasound to a focused region of the hydrogel scaffold with sufficient energy to cavitate the microbubbles causing an uptake of the one or more nucleic acid molecules by at least one target cell from the plurality of target cells present in proximity to the cavitated microbubbles within the focused region of the hydrogel scaffold; and (c) imaging the ultrasound-exposed hydrogel scaffold to visualize the at least one target cell that comprises the one or more nucleic acid molecules; wherein the at least one target cell expressing the one or more nucleic acid molecules is the transgenic target cell.

50. The method of claim 49, wherein the hydrogel scaffold is bioprinted coaxially.

51. The method of claim 49 or claim 50, wherein the hydrogel scaffold comprises a bioink selected from the group of alginate, fibrin, agarose, chitosan, gellan gum, cellulose, hyaluronic acid, gelatin methacryloyl, and methacrylated collagen, or any combinations thereof.

52. The method of claim 51, wherein the hydrogel scaffold comprises alginate bioink.

53. The method of claim 52, wherein the alginate bioink is printed with a printing pressure ranging from about 10 kPa to about 15 kPa.

54. The method of any of claims 49-53, wherein each microbubble of the population of ultrasound-responsive microbubbles comprises an envelope and a gas core.

55. The method of claim 54, wherein the envelope of each microbubble comprises a lipid monolayer or multilayer.

56. The method of claim 54, wherein the gas core of each microbubble comprises a perfluorocarbon gas.

57. The method of claim 56, wherein the perfluorocarbon gas further comprises a gas selected from the group of CF4, C2F4, C2F6, C3F6, C3F8, C4F8, and C4F10, or any combinations thereof.

58. The method of any of claims 49-57, wherein each microbubble has a diameter ranging from about 0.05 μm to about 5 μm.

59. The method of any of claims 49-58, wherein the one or more nucleic acid molecules bound to the microbubbles are selected from the group of a linear DNA, circular DNA, cDNA, miRNA, shRNA, siRNA, plasmid, viral construct, and transposase construct, or any combinations thereof.

60. The method of any of claims 49-59, wherein the one or more nucleic acid molecules are bound to the microbubbles via electrostatic interaction.

61. The method of any of claims 49-60, wherein a distance between the target cells and the microbubbles within the filament structures of the hydrogel scaffold ranges from about 0 μm to about 35 μm.

62. The method of any of claims 49-61, wherein applying ultrasound comprises applying an ultrasonic frequency ranging from about 0.5 MHz to about 15 MHz.

63. The method of claim 62, wherein applying ultrasound further comprises an application of a plurality of ultrasound pulses.

64. The method of claim 63, wherein the plurality of ultrasound pulses ranges from about 10 pulses to about 80 pulses.

65. The method of claim 64, wherein a length of each ultrasound pulse of the plurality of ultrasound pulses ranges from about 0.1 ms to about 100 ms.

66. The method of any of claims 49-65, wherein a size of the focused region where ultrasound is applied within the hydrogel scaffold ranges from about 0.3 mm to about 2 mm.

67. The method of any of claims 49-66, wherein the target cells comprise mammalian single cells or cell aggregates dispersed throughout the filament structures of the hydrogel scaffold.

68. The method of any of claims 49-67, wherein the uptake of the one or more nucleic acid molecules by at least one target cell within the focused region of the hydrogel scaffold is caused by an inertial cavitation of the microbubbles upon ultrasound application.

69. The method of any of claims 49-68, wherein imaging the ultrasound-exposed hydrogel scaffold comprises visualizing and measuring a diameter of a zone comprising the transgenic target cells along the filament structures.

70. A composition prepared by a process comprising the steps of: (a) fabricating a bioink for coaxial bioprinting; (b) incorporating a plurality of microbubbles into the fabricated bioink; (c) incorporating a population of mammalian cells into the fabricated bioink comprising the microbubbles; (d) bioprinting the fabricated bioink comprising the microbubbles and the mammalian cells to form a construct with a plurality of filament structures; and(e) bioprinting a collagen cast gel embedding the plurality of filament structures of the construct to form the composition.

71. The composition of claim 70, wherein the bioink comprises alginate.

72. The composition of claim 71, wherein the alginate is used at a concentration of from about 2% to about 6% weight by volume.

73. The composition of claim 70 or claim 71, wherein the alginate bioink is printed with a printing pressure ranging from about 10 kPa to about 15 kPa.

74. The composition of claim 70, wherein the bioink comprises gelatin.

75. The composition of claim 74, wherein the gelatin bioink is printed with a printing pressure ranging from about 40 kPa to about 60 kPa.

76. The composition of any of claims 70-75, wherein bioprinting the fabricated bioink further includes an addition of a crosslinking agent.

77. The composition of any of claims 70-76, wherein the microbubbles are ultrasound responsive.

78. The composition of any of claims 70-77, wherein the mammalian cells comprise single cells or cell aggregates dispersed throughout the filament structures of the construct.

79. The composition of any of claims 70-78, wherein the microbubbles are dispersed throughout the filament structures of the construct and are located in close proximity to the mammalian cells within the construct.

80. The composition of claim 79, wherein a distance between the mammalian cells and the microbubbles within the filament structures of the construct ranges from about 0 μm to about 35 μm.

81. The composition of any of claims 70-80, wherein the microbubbles are coupled to a substance selected from the group of a nucleic acid, peptide, polypeptide, amino acid, protein, and drug, or any combinations thereof.

82. The composition of any of claims 70-81, wherein the collagen cast gel is selected from the group of type I collagen, type II collagen, and type III collagen, or any combinations thereof.

83. The composition of any of claims 70-82, wherein each filament structure of the plurality of filament structures has a size ranging from about 150 μm to about 700 μm in diameter.

84. The method of claim 49, wherein the hydrogel scaffold is bioprinted in freeform.

85. The method of claim 49 or claim 84, wherein the plurality of filament structures of the hydrogel scaffold comprises a hollow bifurcated construct.

86. The method of claim 84 or claim 85, wherein the hydrogel scaffold is bioprinted in a support bath comprising a slurry of gelatin beads.

87. The composition of any of claims 1-3, wherein the plurality of filament structures of the hydrogel scaffold comprises a planar geometry.

88. The composition of claim 1, wherein the plurality of filament structures of the hydrogel scaffold comprises a freestanding multilayer volumetric geometry.

89. The composition of claim 88, wherein the freestanding multilayer volumetric geometry further comprises a structure selected from the group of a hollow branched structure, interlocking rings, dumbbells, complex pore network, spiral structure, and nested configuration, or any combinations thereof.

90. The composition of claim 89, wherein the freestanding multilayer volumetric geometry comprises the hollow branched structure.

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