System for creating human tissue samples through three-dimensional bioprinting and culturing

Abstract

This document relates to devices, systems, and methods for fabricating a three-dimensional sample of human tissue. For example, this document relates to techniques for depositing cells according to a predefined pattern and treating the deposited cells to simulate a realistic human tissue sample.

Description

Attorney Docket No : 07039-2350W01 / 2023-339 SYSTEM FOR CREATING HUMAN TISSUE SAMPLES THROUGH THREE- DIMENSIONAL BIOPRINTING AND CULTURINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Application No. 63 / 740.718, filed on December 31, 2024, the contents of which are hereby incorporated by reference.TECHNICAL FIELD

[0002] This document relates to devices, systems, and methods for fabricating a three-dimensional sample of human tissue. For example, this document relates to techniques for depositing cells according to a predefined pattern and treating the deposited cells to simulate a realistic human tissue sample.BACKGROUND

[0003] Creating a tissue sample through three-dimensional (3D) bioprinting can involve preparing a bioink including living cells mixed with biocompatible hydrogel precursors. This bioink can be deposited layer-by-layer to create a cellular mass that mimics a desired tissue architecture, guided by a digital model. The cellular mass can be placed in a bioreactor or culture environment promoting cell growth through nutrients, oxygen, and other grow th factors. Over time, the cells can proliferate, differentiate, and interact within the cellular mass, forming a functional tissue sample that closely resembles natural tissue in structure and function.SUMMARY

[0004] This document relates to devices, systems, and methods for fabricating a three-dimensional sample of human tissue. For example, this document relates to techniques for depositing cells according to a predefined pattern and treating the deposited cells to simulate a realistic human tissue sample. In one example, the human tissue sample can be a realistic human skin sample that mimics conditions of ordinary skin samples (e.g., characteristics brought through aging). To create a realistic human tissue sample, a system can create a dermis layer and an epidermis layer. The system can create the dermis layer by depositing a bioink in a layer-by-layer manner. This dermis layer can be exposed to a fibroblast growth medium to promote dermal cell growth for a period of time. Next, the dermis layer can be seeded and / or printed with epidermal cells and exposed to aAttorney Docket No : 07039-2350W01 / 2023-339 keratinocyte growth medium, this time to promote epidermal cell growth. These seeded epidermal cells can grow into an epidermis layer on top of the dermis layer, just like the anatomy of genuine human skin.

[0005] In some examples, the dermis consists of fibroblasts and immune cells and the epidermis consists of melanocytes and keratinocytes. It is possible to formulate dermal bioinks by suspending fibroblasts and collagen hydrogel precursors in aqueous buffer solutions. In some cases, it is beneficial to formulate melanocyte-epidermal bioinks by suspending melanocytes and collagen hydrogel precursors in aqueous buffer solutions. In some embodiments, it can be beneficial to bioprint dermal bioinks and then seed and / or print keratinocytes. In other embodiments, it is beneficial to bioprint dermal bioinks followed by melanocyte-epidermal bioinks, subsequently seeding keratinocytes.Keratinocytes can be suspended in keratinocyte grow th medium and then seeded, in some cases.

[0006] In one aspect, a method comprises depositing a bioink in a layer-by-layer manner to create a three-dimensional dermis layer of cellular matter. The bioink comprises dermal fibroblasts suspended in an aqueous buffer solution. The method also comprises exposing the three-dimensional mass dermis layer of cellular matter to a fibroblast growth medium for a first period of time and, after the first period of time, seeding the three-dimensional dermis layer of cellular matter with epidermal keratinocytes. Additionally, the method comprises exposing the seeded three-dimensional dermis layer of cellular matter to a keratinocyte growth medium for a second period of time. After the second period of time, the method comprises mounting the seeded three-dimensional mass of cellular matter on an insert until the seeded epidermal keratinocytes form a three-dimensional epidermis layer of cellular matter on top of the three-dimensional dermis layer of cellular matter.

[0007] Particular embodiments of the subject matter described in this document can be implemented to realize one or more of the following advantages. For example, placing a tissue sample in a fibroblast growth medium to promote dermis cell growth before seeding the dermis layer with epidermis cells can cause the tissue sample to have characteristics associated with genuine tissue samples. For example, the fibroblast growth medium can cause the cells to interact with one another in a way that is expected in real tissue samples. Furthermore, seeding the dermis layer with epidermal cells and mounting the seeded dermis on an insert can cause the epidermis to develop in the same w ay that the human epidermis develops in nature. For example, the insert can suspend the tissue sample in a media so that part of the sample is exposed to air and part of the sample is exposed to aAttorney Docket No : 07039-2350W01 / 2023-339 differentiation media. This simulates the conditions of human skin to promote development of the epidermis.

[0008] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0009] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description herein. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 A illustrates a first example system with keratinocytes and fibroblasts for creating tissue skin samples including a dermis layer and an epidermis layer.

[0011] FIG. IB illustrates a second example system with keratinocytes, melanocytes, and fibroblasts for creating tissue skin samples including dermis and epidermis layers.

[0012] FIG. 1C illustrates a third example system with keratinocytes and fibroblasts for creating tissue skin samples having induced radiation dermatitis, the samples including a dermis layer and an epidermis layer.

[0013] FIG. 2 illustrates a set of dermis layers deposited by a 3D bioprinter.

[0014] FIGS. 3A-3K illustrate examples of an insert that can rest in a culture dish as part of a process for creating tissue samples.

[0015] FIG. 4 illustrates a process for creating polyacrylamide hydrogels.

[0016] FIGS. 5A-5D illustrate patient-specific spatial mapping for use in a senescence biological aging model.

[0017] FIG. 6 is a flow diagram illustrating how a print head and a seeding device can deposit material to fabricate a three-dimensional sample of human tissue.DETAILED DESCRIPTION

[0018] This document relates to devices, systems, and methods for fabricating a three-dimensional sample of human tissue as depicted in FIG. 1A. For example, this documentAttorney Docket No : 07039-2350W01 / 2023-339 relates to techniques for depositing cells according to a predefined pattern and treating the deposited cells to simulate a realistic human tissue sample.

[0019] In some embodiments, a system can create three-dimensional (3D) skin tissue samples using 3D bioprinting technology and bioinks formulated with pre-polymers of collagen hydrogels and primary human cell lines. In some cases, it is beneficial to create tissue samples that approximate conditions of genuine tissue samples found in nature. Human skin can have different characteristics depending on a number of factors such as age and exposure to sunlight, among others. Creating tissue samples that mimic characteristics of human skin can provide scientists w ith a way to generate realistic tissue samples for scientific studies and manufacture tissue samples for use in skin grafts on human patients to treat wounds and disease, among other uses.

[0020] Bioinks can consist of cells and hydrogel precursors (hydrogel monomer molecules) suspended in aqueous buffer solutions. Hydrogels can be natural or synthetic polymers which provide scaffold support to cells. It is possible to use hydrogel monomer molecules in bioinks. After deposition these hydrogel monomer molecules can assemble, crosslink, and polymerize into hydrogel. It is possible to use two types of collagen monomer molecules. The first is animal-derived collagen monomer molecules such as bovine collagen monomer molecules. These can crosslink into collagen hydrogel at 37° Celsius. Alternatively, these monomer molecules can be chemically modified to crosslink into collagen hydrogel when exposed to UV light. The second is recombinant human collagen monomer molecules derived from tobacco plant leaves. These crosslink into collagen hydrogel when exposed to UV light.

[0021] Human lifespan continues to increase because of the advances in medicine.However, a quality of life or health span of older individuals can suffer due to progressive decline in their physiological functions associated with accumulation of damage at the cellular level with their age. Examples of such damage include genetic mutations, reduced telomere length, misfolded proteins, impaired nutrient metabolism, cellular senescence, and stem cell exhaustion. Conditions such as cancer, musculoskeletal disorders, neurodegenerative diseases such as Alzheimer’s, cardiovascular diseases such as atherosclerosis, and autoimmune diseases such as allergy can result from tissue damage. Skin tissue can be adversely affected with age. For example, compromised skin barrier functionality can be associated with chronic inflammatory diseases and autoimmune diseases even at a younger age. In addition, accumulation of genetic mutations in the skin with age can cause malignant cancers which spread to other tissues in the human body.Attorney Docket No : 07039-2350W01 / 2023-339

[0022] Skin can be ideal for studying aging because skin is susceptible to damage induced by environmental factors such as sun exposure (ultraviolet radiation) and intrinsic factors such as impaired nutrient metabolism by mitochondria. Aging and aging-related diseases have been studied with two-dimensional (2D) monolayer cell cultures and animal experimentation platforms. Two-dimensional monolayer cell cultures reveal valuable but limited information on aging since they do not replicate three-dimensional (3D) structure of the tissues and do not facilitate complex intercellular crosstalk among different cell types in vitro. Animals can differ humans in form and function. For example, sweat glands are absent in murine skin and its renewal occurs every 8 - 10 days in comparison to 28 days of the human skin. This means that it can be preferable to generate human tissue samples for scientific studies instead of using animal skin samples or animal testing.

[0023] A skin tissue sample can include, in some examples, fibroblasts and keratinocytes. Fibroblasts are a kind of cell in connective tissue and can play a crucial role in maintaining a structural framework of tissues by producing extracellular matrix components such as collagen, elastin, and glycosaminoglycans. Fibroblasts can be spindle-shaped and versatile, capable of responding to various stimuli, such as growth factors or mechanical stress, to modify their activity. Fibroblasts can contribute to wound healing by migrating to the site of injury, proliferating, and synthesizing new matrices to repair damaged tissue. They also interact with other cell types, like immune cells, to coordinate tissue regeneration and repair processes.

[0024] In the dermis layer of the skin, fibroblasts can maintain the structural and functional integrity of the skin by producing and remodeling extracellular matrix components, such as collagen and elastin, which provide strength and elasticity.Fibroblasts play a role in skin homeostasis, responding to signals from surrounding cells and environmental cues to regulate matrix turnover and repair. During wound healing, fibroblasts become activated, proliferating and migrating to the site of injury, where fibroblasts can synthesize new extracellular matrix to replace damaged tissue. Fibroblasts can also interact with immune cells to mediate inflammation and secrete growth factors that promote keratinocyte proliferation and angiogenesis. Dysregulation of fibroblast activity in the dermis can lead to conditions such as scarring, fibrosis, or impaired wound healing.

[0025] Keratinocytes are predominant cell in the epidermis, the outermost layer of the skin. These cells are responsible for forming a protective barrier against environmental damage, such as pathogens, ultraviolet (UV) radiation, and water loss. KeratinocytesAttorney Docket No : 07039-2350W01 / 2023-339 originate from basal cells in the stratum basale, where they undergo proliferation. As keratinocytes migrate upward through the epidermal layers, they differentiate, producing keratin and lipid-based molecules that contribute to the skin's mechanical strength and water-retention properties. Ultimately, these cells reach the stratum comeum, where they become enucleated comeocytes, forming a durable, protective layer.

[0026] Keratinocytes are also active participants in skin homeostasis and immune response. They secrete cytokines and growth factors that influence inflammation and wound healing, working in concert with immune cells to defend against infections. When the skin is damaged, keratinocytes rapidly proliferate and migrate to close the wound, supported by signals from fibroblasts and other cell types. Dysregulation of keratinocy te function can lead to skin diseases such as psoriasis, eczema, and skin cancer, emphasizing their pivotal role in both skin integrity and overall health.

[0027] To create a tissue sample that is similar to genuine human skin, the system can create a dermis layer where fibroblasts are prevalent and an epidermis layer where keratinocytes and / or melanocytes are prevalent. In some cases, synthetic polymers such as polyacrylamide hydrogels can be used as two-dimensional (2D) culture substrates to identify an elastic modulus that enhances biological functions of primary dermal fibroblasts, epidermal keratinocytes, epidermal melanocytes, or any combination thereof.3D bioprinted tissue samples have several advantages that are not present in 2D culture monolayer cultures, including realistic dermis and epidermis layers that simulate characteristics of genuine skin. 3D bioprinting emulates important physiological features of human tissues including skin in a dish by delivering living cells, biopolymers, and biological molecules at predetermined spaces. In some cases, bioprinted 3D tissues can replace two-dimensional (2D) monolayer cultures and animal experimentation platforms during novel pharmaceutical drug development to minimize a failure of drugs during human clinical trials. Because monolayer cultures do not emulate 3D microenvironments of the human body, bioprinted 3D tissue samples can provide characteristics that are found in these environments.

[0028] Furthermore, bioprinting can facilitate high-throughput manufacturing of individualized 3D human tissues representing diverse demographics for development of diagnostic and therapeutic interventions. For example, skin samples can be created to simulate skin of juveniles, young adults, older adults, or any combination thereof. Skin characteristics can also differ according to other factors such as sex, race, and level of exposure to sunlight. It can be beneficial to create samples that simulate thoseAttorney Docket No : 07039-2350W01 / 2023-339 characteristics. Bioprinting can be advantageous because many different skin samples can be created according to specific demographic characteristics that are the subject of a given scientific study. For example, many samples simulating the skin of older subjects can be created for a study focusing on treatments for skin conditions common in older patients.

[0029] According to one example process for creating tissue samples to simulate tissue samples of skin in older patients, fibroblasts from young donors can be used after artificially inducing senescence in the fibroblasts. According to another example process, keratinocytes and fibroblasts from older donors (e g., 70 years old) can be used to create tissue samples without inducing senescence. One limitation of these young and old skin models is that skin models can be created by manually casting cell-laden hydrogel precursor solutions onto porous membranes. This does not provide control over the tissue geometry or placement of skin cells at predetermined spaces. This approach of manually casting onto porous membranes can be susceptible to human error. The system described herein uses 3D bioprinting and seeding of keratinocytes to create tissue samples in a way that does not rely on the error-prone placement of cells into porous membranes or in specific locations on a scaffold.

[0030] 3D bioprinting is an automated-robotic technology which places living cells, biopolymers, and biological signaling molecules at predetermined spaces to mimic essential features of skin in a dish, including advanced aging. In some cases, young human 3D skin models can be bioprinted using precursors of alginate, collagen, fibrin, and gelatin hydrogels as bioink formulations. The stiffness or elastic modulus of an extracellular matrix (ECM) can control cell fate and functions, and each cell type corresponding to a particular stiffness where cell functions are enhanced. An ideal ECM stiffness for human skin can be difficult to determine since measurement of human skin stiffness has been inconsistent, with stiffness ranging from under 1 kilopascal (kPa) to hundreds of megapascals (MPa) in several studies. In some cases, the system described herein can bioprint young and old 3D human skin models using primary epidermal keratinocytes and primary dermal fibroblasts from young and old donors, respectively. The system can also identify ideal 3D scaffold stiffness that enhances functioning of the two cell types for the two specific age groups.

[0031] Compromised barrier functionality7can be associated w ith the onset of chronic inflammatory diseases such as atopic dermatitis and autoimmune diseases such as allergy and asthma. Skin is most adversely affected with age and accumulation of skin damage at the cellular level with time is putting older individuals at the increased risk of chronic skinAttorney Docket No : 07039-2350W01 / 2023-339 wounds and diseases such as malignant skin cancer, diminishing their quality of life despite the increase in their lifespan. Bioprinted human 3D is an alternative to animal experimentation for developing diagnostic and therapeutic interventions. Some systems for creating bioprinted 3D tissue samples rely on manual seeding of primary human dermal fibroblast (NHDF) cells into porous plastic membranes or scaffolds or rely on plant-derived recombinant human collagen scaffolds. In some cases, techniques that involve manual seeding into a porous scaffold does not offer control over the placement of cells at predetermined spaces and is susceptible to human errors, limiting high-throughput manufacturing of the skin as well as high-throughput screening of diagnostic and therapeutic interventions. The system described herein can generate bioprinted 3D tissue samples without relying on manual seeding into porous scaffolds and therefore decreasing a likelihood of human error.

[0032] In some examples, bioprinting can be used to create 3D skin equivalents with NHDF cells, primary epidermal keratinocyte (NHEK) cells, and animal-derived type I collagen. However, animal-derived collagen can in some cases induce elevated immunological response. In some cases, the system described herein can use bioprinting to create fully humanized 3D skin with plant-derived recombinant human type I collagen, NHDFs, primary human epidermal melanocyte (NHEM) cells, and NHEKs. This plant-derived collagen can be a photocrosslinkable variant of a collagen used to create a fully humanized 3D skin with NHDFs. NHEKs, and primary’ human endothelial cells.

[0033] Collagen bioinks con be formulated to yield hydrogels, which mimic an ECM for bioprinted cells, with an elastic modulus of an ECM. For example, a collagen type I hydrogel precursor solution can be used as a constituent bioink formulation to provide 3D scaffold support to bioprinted skin cells. Collagen is a chief component of the ECM in human tissues and better mimics physiological microenvironments. The stiffness of the collagen 3D scaffold has been modulated by vary ing the concentration collagen monomer molecules in the precursor solution. Furthermore, polyacry lamide hydrogels with vary ing stiffness have been used as 2D culture substrates. These hydrogels can be synthetic hydrogels and adopted as a standard platform for studying the impact of stiffness on cellular behavior.

[0034] Polyacrylamide hydrogels with five different controlled stiffness values can be used for identifying an elastic modulus or mechanical stiffness of 2D culture surfaces and 3D scaffolds at which biological functions of cells including skin cells is improved. These hydrogels can be synthesized according to previously published protocols. Briefly, 18-mmAttorney Docket No : 07039-2350W01 / 2023-339 glass coverslips can be treated with 0.1 N sodium hydroxide (NaOH) solution for 5 minutes. The NaOH solution can be aspirated, and the coverslips can be treated with 97% (3-Aminopropyl)trimethoxysilane (3-APTMS, Sigma- Aldrich, Cat # 281778 ) for 5 minutes. The reagent can be removed by aspiration and the coverslips can be washed three times with Milli-Q water, each wash lasting 10 minutes. The washed coverslips can be dried by aspiration and treated with 0.5% glutaraldehyde solution for 30 minutes. The reagent can be removed by aspiration and the coverslips were washed three times with Milli-Q water, each wash lasting 10 min. The washed coverslips can be dried by aspiration and were set aside for further air-drying. The air-dried coverslips can be referred to as reactive coverslips.

[0035] In some embodiments, polyacrylamide hydrogels can be synthesized by preparing a pre-polymer solution containing following reagents, Milli-Q water, acrylamide solution, bis-acrylamide, 10% w / v ammonium persulfate solution, saturated acrylic acid N-hydroxy-succinimide ester solution, up to 55 mg / mL in Milli-Q water, N, N, N’, N’-tetramethylethylenediamine. A specific composition of a pre-polymer solution for each stiffness value can be listed under Table 1. The pre-polymer solution can be briefly vortexed and 120 pL of the solution can be pipetted on to a reactive coverslip.Immediately afterwards, an 18-mm siliconized coverslip (Greiner, Hampton Research, Aliso Viejo, CA, Cat # HR3-255) can be placed on the reactive coverslip. The pre-polymer solution can be allowed to polymerize for 10 minutes for 0.7, 3, and 10 kPa hydrogels whereas 20 min for 25 and 50 kPa hydrogels. After polymerization, the siliconized coverslip can be removed and the reactive coverslip with the hydrogel can be transferred to anon-treated 12-well plate containing Dulbecco’s phosphate-buffered saline (DPBS) without calcium and magnesium, IX concentration.Table 1

[0036] In some embodiments, Polyacrylamide hydrogels do not facilitate attachment of cells unless they are covalently functionalized with ECM molecules. Hence, theseAttorney Docket No : 07039-2350W01 / 2023-339 hydrogels can be functionalized with recombinant human laminin-332 E8 fragments (iMatrix-332, Nippi Inc., Tokyo, Japan. Cat # 892032) to facilitate the attachment of keratinocytes. Similarly, the hydrogels were functionalized with recombinant human collagen type I molecules (Collage, CollPlant Ltd., Rehovot, Israel, Cat # W1019) to facilitate the attachment of fibroblasts. To functionalize and to control the orientation of laminin-332 fragments, the hydrogels can be treated with 5 mMN-(2-aminoethyl)maleimide trifluoro acetate at pH 7.5 for 2 h at room temperature. Afterwards, the hydrogels can be washed once with 70% ethanol and three times with DPBS to sterilize them. The sterilized hydrogels can be transferred to new non-treated 12-well plates and were treated with the laminin-332 fragment at a density of 5 pg / mL in DPBS without calcium and magnesium for overnight at 4 °C. The following day, the hydrogels can be washed twice with DPBS, 2 mL of keratinocy te grow th medium being added to each well, and keratinocytes can be seeded at a density of 50,000 cells / cm2on each hydrogel. The cells can then be cultured in the humidified 5% CO2 incubator at 37 °C for 48 h. The hydrogels can be directly sterilized as described before. The sterilized hydrogels can be treated with collage at a density of 100 pg / mL in DPBS without calcium and magnesium at pH 7.5 for overnight at room temperature. The following day, the hydrogels can be washed twice with DPBS, 2 mL of fibroblast grow th medium w as added to each well, and fibroblasts can be seeded at a density of 12,500 cells / cm2on each hydrogel. The cells can then be cultured in the humidified 5% CO2 incubator at 37 °C for 48 h.

[0037] In some examples, a bioink for creating a dermis layer of a skin tissue sample can be created using NHDF cells, sometimes referred to as “dermal fibroblasts.” To create a bioink, collagen ty pe I monomer molecules and dermal NHDF cells can be suspended in an aqueous buffer solution so that the collagen type I monomer molecules have a predetermined concentration and the NHDF cells have a predetermined concentration. In some examples, the concentration of the NHDF cells is within a range from 2 X 104- milhleter ( —mL) - to 40 x 106mL and the concentration of the collagen tvpe I monomer molecules is within a range from 2mMiarams ('m9)(040 212mL mL

[0038] In some examples, a dermal bioinks can be formulated using a plant-derived recombinant human type I collagen methacrylamide (15.5Collink.3D 50, CollPlant, Cat # W10199). To neutralize the stock solution of collagen methacylamide. in some embodiments, for every 1 gram (g) of the solution, 15.2 microleters (ji ) of 1 normalAttorney Docket No : 07039-2350W01 / 2023-339 (N) sodium hydroxide (NaOH) and 102 pL of 10 times concentrated stock solution (10X) Dulbecco’s phosphate-buffered saline (DPBS) without calcium and magnesium can be added. The neutralized solution can be diluted to a desired concentration of collagen using IX DBPS without calcium and magnesium. In some cases, a stock solution of 2% w / v lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) can be prepared using IX DPBS. The LAP solution can be added to the diluted collagen solution. The dermal bioink can be formulated by resuspending 200,000 NHDF cells in 50 pL of fibroblast growth medium and adding this growth medium to 1 mL of the collagen / LAP solution, obtaining a bioink including collagen according to a predetermined concentration. In some cases, the collagen concentration can be within a range from 5 — mL to 40 — mL , within a range from 5 — mL tomq10 — , or within another range. In some examples, a concentration of LAP in the bioink is • 1 •within a range from

[0039] In some embodiments, an epidermal bioink can be formulated by suspending 2 x 104- millileter - (mL) to 40 x 106mL NHEM cells (sometimes referred to as "‘epidermal melanocytes”) in 50 pL of melanocyte growth medium and adding this medium to I mL of the collagen / LAP solution used to create the dermal bioink. This .u epidermal bioink can, in some examples, have a collagen concentration value of 5 — and a final LAP concentration value of 0.15% Additionally, or alternatively, NHEK cells (sometimes referred to as "epidermal keratinocytes”) can be mixed with keratinocytes growth medium to create an epidermal bioink.

[0040] In some embodiments, NHDF cells, NHEMs cells, and NHEK cells can be cultured in their respective growth mediums in a humidified 5% carbon dioxide (CO2) incubator at 37 °C. In some cases, media can be changed once in 2 - 3 days. Confluent cells can be detached from culture flasks by incubating with 0.05% trypsin solution for 5 minutes. After 5 minutes, the trypsin solution can be neutralized with Dulbecco’s modified eagle medium (DMEM) including 10% fetal bovine serum. The neutralized solution can be centrifuged at 1800 rotations per minute (rpm) for 5 minutes. The obtained cell pellet can be resuspended in an appropriate growth medium, and cells can be transferred to new culture flasks for further expansion. Alternatively, a cell pellet can be used for bioprinting experiments.Attorney Docket No : 07039-2350W01 / 2023-339

[0041] A print head 10 of a 3D bioprinting device can be used, in some examples, to deposit a dermal bioink in a layer-by-layer manned to create a three-dimensional dermis layer of cellular matter 20. As depicted in FIG. 1 A, the three-dimensional dermis layer of cellular matter 20 includes a set of layers 12 stacked on top of one another upwards from a substrate. The 3D bioprinting device can control a position of the print head 10 to deposit each of these layers 12 according to a predefined pattern. The predefined pattern for each of the layers can be determined based on a 3D model (e.g.. a computer assisted design (CAD) model) of the dermis layer. Depositing the layers 12 to create the three-dimensional dermis layer of cellular matter 20 may, in some examples, occur at time Tl. Upon deposition, the dermal bioink can, in some examples, polymerize into collagen hydrogel within a period of time (e.g., within a range from 3 to 5 minutes). In some embodiments, the dermal bioink can polymerize into an animal-derived collagen within a range from three to five minutes after deposition at a predetermined temperature (e.g., 37° Celsius), and subsequently begin to crosslink. In some embodiments, the dermal bioink can polymerize into a plant-derived collagen which crosslinks immediately when exposed to light at a predetermined wavelength (e.g., 405 nanometers (nm)).

[0042] In some embodiments, humanized 3D skin tissues can be bioprinted with a 3D bioprinting device that is able to extrude a dermal bioink. Before using the 3D bioprinting device to extrude the dermal bioink, the dermal bioink (e.g., a dermal bioink having 5collagen and 0.15 %LAP) can be transferred to a sterile cartridge (e.g., a 3 mL cartridge). A blunt needle (e.g., a 30 gauge (G) blunt needle) can be attached to the cartridge and maintained at a predetermined temperature (e.g., 24 °C) for feeding to the 3D bioprinting device. In some cases, the printhead 10 of the 3D bioprinting device can generate one or more dermis constructs measuring 12.5 mm x 12.5mm x 3 mm. These dermis constructs can be bioprinted and photo-cured at 405 nanometers (nm) for a period of time (e.g., 30 seconds) following deposition of the dermal bioink.

[0043] In some embodiments, three-dimensional skin models can be bioprinted using a 3D bioprinting device that is able to extrude a dermal bioink. Confluent fibroblasts can be detached from culture flasks and resuspended in a fibroblast growth medium at a(i cellspredetermined density (e.g., 4 x 10 y^y). A growth medium containing cells can be mixed with bovine collagen type I hydrogel precursor solutions. In some cases, for every 1 mL of neutralized collagen solutions, 50 pL of the grow th medium containing the cells can be added. Specific bioink formulations are listed below under Table 2. Each collagenAttorney Docket No : 07039-2350W01 / 2023-339 solution can be transferred to a sterile cartridge (e.g., a 3 mL cartridge) and a blunt needle (e.g., a 30G blunt needle) can be attached to the cartridge. The temperature of the cartridge can be maintained at a predetermined temperature (e.g., 4 °C) inside the bioprinter. The 3D bioprinting device can use the print head 10 to print one or more 10mm x 10mm x 3 mm dermis constructs. In some cases, the bioprinted dermis constructs can be incubated for a period of time (e.g., within a range from 60 minutes to 90 minutes) in a humidified 5% CO2 incubator at a predetermined temperature (e.g., 37 °C) to facilitate crosslinking of collagen precursor solution into collagen hydrogel. After the period of time, fibroblast grow th medium can be added to the dermis constructs.Table 2

[0044] When the dermal bioink polymerizes after print head 10 deposits layers 12 of the dermal bioink into a three-dimensional dermis layer of cellular matter 20, the three-dimensional dermis layer of cellular matter 20 can be cultured in a fibroblast growth medium for a period of time. In some examples, this period of time extends from time T1 to time T2. There may be, in some cases, five days between time T1 and time T2, but this is not required. The amount of time between T1 and T2 can be any other amount of time. In some examples the three-dimensional dermis layer of cellular matter 20 can be cultured in the fibroblast growth medium in a humidified incubator at a predetermined temperature (e.g., 37 °C). In some examples, a fibroblast growth medium for the cells can be changed every 2 or 3 days. When cells are confluent, the cells can be detached from culture flasks by incubating the cells with a 0.05% trypsin solution for 5 minutes. The try psin solution can be neutralized with DMEM containing 10% fetal bovine serum. The neutralized trypsin solution containing the cells can be centrifuged at 1800 rpm for 5 minutes to obtain a cell pellet. The pellet can be resuspended in appropriate growth medium, and the cells can be transferred to new culture flasks to facilitate further proliferation or used for 2D monolayer culture experiments. Alternatively, the pellet can be used for bioprinting human 3D skin.Attorney Docket No : 07039-2350W01 / 2023-339

[0045] At time T2, in some examples, a surface of the three-dimensional dermis layer of cellular matter 20 can be seeded with primary’ epidermal keratinocytes at a predetermineddensity' (yvithin a range from 50,000 to 400,000e.g 300.000 ^^). In someembodiments, at time T2, the print head 10 can bioprint one or more layers of the epidermal keratinocytes onto the three-dimensional dermis layer of cellular matter 20 instead of seeding. In some cases where keratinocytes are seeded, confluent keratinocytes can be trypsinized from culture flasks and resuspended in the keratinocyte growth medium at a density (e.g., 3 x io7In some embodiments, the fibroblast culture medium canbe removed and 10 pL of keratinocyte growth medium containing the keratinocytes can be added on each dermis construct. That is, a densify of 300,000 keratinocytes per cm2of surface area can be seeded on each dermis construct. The seeding can be performed by a seeding device 18 that is configured to deposit epidermal keratinocytes on the surface of the three-dimensional dermis layer of cellular matter 20.

[0046] In some examples, the three-dimensional dermis layer of cellular matter 20 can be fabricated using a scaffold, such as a collagen-rich matrix populated with dermal fibroblasts. This can recreate a structural and biochemical environment of native dermis. When keratinocytes are seeded onto the surface of the three-dimensional dermis layer of cellular matter 20. the keratinocytes can attach via integrins to extracellular matrix proteins and receive paracrine signals from the underlying fibroblasts in the three-dimensional dermis layer of cellular matter 20. These cues can promote keratinocyte survival, proliferation, and proper polarity, establishing a basal layer analogous to the stratum basale found in natural epidermis layers.

[0047] Keratinocytes can be bioprinted onto the three-dimensional dermis layer of cellular matter 20 in addition to or alternatively to seeding the three-dimensional dermis layer of cellular matter 20 in some embodiments. For example, the 3D bioprinting device can include a print head (e.g., print head 10 or another print head) that can deposit layers of an epidermal bioink on the three-dimensional dermis layer of cellular matter 20.

[0048] A period of time after the seeding or bioprinting (e.g., 60 minutes after seeding or bioprinting), a keratinocyte growth medium can be added to the skin models, and the skin models can be cultured in submerged conditions until time T3. For example, the seeded and / or bioprinted three-dimensional dermis layer of cellular matter 20 can be submerged in the keratinocyte growth medium between Time T2 and time T3. Between time T2 and time T3, the keratinocyte growth medium can facilitate epidermal keratinocyte cell growth ofAttorney Docket No : 07039-2350W01 / 2023-339 the seeded or bioprinted epidermal keratinocyte cells to form a three-dimensional keratinocyte epidermis layer 22 on top of the three-dimensional dermis layer of cellular matter 20. In some examples, a time between T2 and T3 is 2 days. In some examples, a time between T1 and T3 is 7 days.

[0049] At time T3, a tissue sample 24 including the three-dimensional keratinocyte epidermis layer 22 and the three-dimensional dermis layer of cellular matter 20 can be placed into an insert 30 within a culture dish. The culture dish can be partially filled with a keratinocyte differentiation medium so that a liquid / air interface exists in the insert. For example, the liquid / air interface can exist so that the tissue sample 24 resting on the surface of the insert is partially submerged in liquid and partially exposed to air. This can cause the seeded and / or bioprinted keratinocytes to self-organize and differentiate into a stratified epidermis. In some examples, the tissue sample 24 can be complete at time T4 within a period of 5 days after time T3 when the sample is placed in the insert.

[0050] As culture conditions are adjusted, such as commonly by raising the tissue to an air-liquid interface — the keratinocytes begin to stratify and differentiate. Limited access to nutrients and exposure to air trigger a well-orchestrated program of terminal differentiation, leading to the formation of suprabasal layers and, eventually, a cornified outer layer. Through cell-cell junction formation, calcium signaling, and regulated gene expression, the keratinocytes organize into a multilayered epidermis that mirrors the architecture and barrier function of native skin. This process demonstrates how appropriate cellular context and environmental cues can drive self-organization in fabricated tissue samples.

[0051] Referring now' to FIG. IB, the print head 10 of a 3D bioprinting device can be used, in some examples, to deposit a dermal bioink in a layer-by-layer manner to create a three-dimensional dermis layer of cellular matter 20. As depicted in FIG. IB, the three-dimensional dermis layer of cellular matter 20 includes a set of layers 12 stacked on top of one another upw ards from a substrate. The 3D bioprinting device can control a position of the print head 10 to deposit each of these layers 12 according to a predefined pattern. The predefined pattern for each of the layers can be determined based on a 3D model (e.g., a computer assisted design (CAD) model) of the dermis layer. Depositing the layers 12 to create the three-dimensional dermis layer of cellular matter 20 may, in some examples, occur at time T*l. Upon deposition, the dermal bioink can polymerize, in some examples, into collagen hydrogel within a period of time (e.g., within a range from 3 to 5 minutes). In some embodiments, the dermal bioink can polymerize into an animal-derived collagenAttorney Docket No : 07039-2350W01 / 2023-339 within a range from three to five minutes after deposition at a predetermined temperature (e.g., 37° Celsius), and subsequently begin to crosslink. In some embodiments, the dermal bioink can polymerize into a plant-derived collagen which crosslinks immediately when exposed to light at a predetermined wavelength (e.g., 405 nanometers (nm)).

[0052] In some embodiments, humanized 3D skin tissues can be bioprinted with a 3D bioprinting device that is able to extrude a dermal bioink. Before using the 3D bioprinting device to extrude the dermal bioink, the dermal bioink (e.g., a dermal bioink having 5collagen and 0.15 % LAP) can be transferred to a sterile cartridge (e.g., a 3 mL cartridge). A blunt needle (e.g., a 30 gauge (G) blunt needle) can be attached to the cartridge and maintained at a predetermined temperature (e.g., 24 °C) for feeding to the 3D bioprinting device. In some cases, the print head 10 of the 3D bioprinting device can generate one or more dermis constructs measuring 12.5 mm x 12.5mm x 3 mm. These dermis constructs can be bioprinted and photo-cured at 405 nanometers (nm) for a period of time (e.g., 30 seconds) following deposition of the dermal bioink.

[0053] In some embodiments, three-dimensional skin models can be bioprinted using a 3D bioprinting device that is able to extrude a dermal bioink. Confluent fibroblasts can be detached from culture flasks and resuspended in a fibroblast grow th medium at ac c&llspredetermined density (e.g., 4 x 1 () ’ — ^-). A growth medium containing cells can be mixed with bovine collagen type I hydrogel precursor solutions. In some cases, for every' 1 mL of neutralized collagen solutions, 50 pL of the grow th medium containing the cells can be added. Each collagen solution can be transferred to a sterile cartridge (e.g., a 3 mL cartridge) and a blunt needle (e.g., a 30G blunt needle) can be attached to the cartridge. The temperature of the cartridge can be maintained at a predetermined temperature (e.g., 4 °C) inside the bioprinter. The 3D bioprinting device can use the print head 10 to print one or more 10mm x 10mm x 3 mm dermis constructs. In some cases, the bioprinted dermis constructs can be incubated for a period of time (e.g., within a range from 60 minutes to 90 minutes) in a humidified 5% CO2 incubator at a predetermined temperature (e.g., 37 °C) to facilitate crosslinking of collagen precursor solution into collagen hydrogel. After the period of time, fibroblast growth medium can be added to the dermis constructs.

[0054] When the dermal bioink polymerizes after print head 10 deposits layers 12 of the dermal bioink into a three-dimensional dermis layer of cellular matter 20, the three-dimensional dermis layer of cellular matter 20 can be cultured in a first growth mediumAttorney Docket No : 07039-2350W01 / 2023-339 (e.g., a fibroblast growth medium) for a period of time. In some examples, this period of time extends from time T*1 to time T*2. There may be, in some cases, three days between time T*1 and time T*2, but this is not required. The amount of time between T* 1 and T*2 can be any other amount of time. In some examples, the three-dimensional dermis layer of cellular matter 20 can be cultured in the first growth medium (e.g., the fibroblast growth medium) in a humidified incubator at a predetermined temperature (e.g., 37 °C). In some examples, a fibroblast growth medium for the cells can be changed every 2 or 3 days. When cells are confluent, the cells can be detached from culture flasks by incubating the cells with a 0.05% trypsin solution for 5 minutes. The try psin solution can be neutralized with DMEM containing 10% - fetal bovine serum. The neutralized trypsin solution containing the cells can be centrifuged at 1800 rpm for 5 minutes to obtain a cell pellet. The pellet can be resuspended in appropriate growth medium, and the cells can be transferred to new culture flasks to facilitate further proliferation or used for 2D monolayer culture experiments. Alternatively, the pellet can be used for bioprinting human 3D skin.

[0055] At time T*2, the print head 10 can bioprint a set of layers 13 of an epidermal bioink comprising epidermal melanocytes on top of the three-dimensional dermis layer of cellular matter 20. These set of layers 13 can represent a three-dimensional melanocyte epidermis layer 21. In some examples, the epidermal layer can have certain dimensions (e.g., 10mm x 10mm x 1mm). The three-dimensional melanocyte epidermis layer 21 can be photo-cured at a predetermined wavelength (e.g., 405nm) for a period of time (e.g., for 30 seconds). After the epidermal layer is printed and photo-cured, the epidermal layer can be cultured in a second growth medium (e.g.. a melanocyte growth medium) for a period of time between time T*2 and time T*3. In some cases, the amount of time between time T*2 and time T*3 is 2 days, but this is not required. The amount of time between time T*2 and time T*3 can be any other amount of time.

[0056] At time T*3, in some embodiments, a surface of the three-dimensional dermis layer of cellular matter 20 can be seeded with primary epidermal keratinocytes at a predetermined density (within a range from 50,000 to 400,000e.g., 300,000In other embodiments, at time T*3, the print head 10 can bioprint one or more layers of a bioink comprising epidermal keratinocytes on top of the three-dimensional melanocyte epidermis layer 21. In some cases where the epidermal keratinocytes are seeded, confluent keratinocytes can be trypsinized from culture flasks and resuspended in the keratinocyte growth medium at a density (e.g., 3 x io7[n SOme embodiments, the fibroblastAttorney Docket No : 07039-2350W01 / 2023-339 culture medium can be removed and 10 pL of keratinocyte growth medium containing the keratinocytes can be added on each dermis construct. That is, a density of 300,000 keratinocytes per cm2of surface area can be seeded on each dermis construct. The seeding can be performed by a seeding device 18 that is configured to deposit epidermal keratinocytes on the surface of the three-dimensional dermis layer of cellular matter 20.

[0057] A period of time after the seeding or bioprinting (e.g., 60 minutes after seeding or bioprinting), keratinocyte growth medium can be added to the skin models, and the skin models can be cultured in submerged conditions until time T*4. For example, the seeded three-dimensional dermis layer of cellular matter 20 and the three-dimensional melanocyte epidermis layer 21 can be submerged in the keratinocyte grow th medium between time T*3 and time T*4. Between time T*3 and time T*4, the keratinocyte growth medium can facilitate epidermal keratinocyte cell growth of the seeded or bioprinted epidermal keratinocyte cells in order to form a three-dimensional epidermis including a three-dimensional melanocyte epidermis layer 21 comprising epidermal melanocytes and a three-dimensional keratinocyte epidermis layer 22 comprising epidermal keratinocytes on top of the three-dimensional dermis layer of cellular matter 20. Together, the three-dimensional dermis layer of cellular matter 20, the three-dimensional melanocyte epidermis layer 21, and the three-dimensional keratinocyte epidermis layer 22 comprise a tissue sample 26 that includes both melanocytes and keratinocytes in the epidermal layers. In some examples, a time between T*3 and T*4 is 2 Days. In some examples, a time between T*1 and T*4 is 7 days.

[0058] At time T*4, the tissue sample 26 including the three-dimensional dermis layer of cellular matter 20, the three-dimensional melanocyte epidermis layer 21, and the three-dimensional keratinocyte epidermis layer 22 can be placed into an insert 30 within a culture dish. The culture dish can be partially filled with a keratinocyte differentiation medium so that a liquid / air interface exists in the insert. For example, the liquid / air interface can exist so that the tissue sample resting on the surface of the insert is partially submerged in liquid and partially exposed to air. This can cause the seeded and / or printed melanocytes and / or keratinocytes to self-organize and differentiate into a stratified epidermis layer comprising the three-dimensional melanocyte epidermis layer 21 and the three-dimensional keratinocyte epidermis layer 22. In some examples, the tissue sample 26 can be completed at time T*5 within a period of five days after time T*4 when the sample is placed in the insert.Attorney Docket No : 07039-2350W01 / 2023-339

[0059] Referring now to FIG. 1C, a three-dimensional dermis layer of cellular matter 20 can be bioprinted by a print head 10 at time T1 and cultured in a fibroblast growth medium between time T1 and time T2. At time T2, the three-dimensional dermis layer of cellular matter 20 can be seeded with keratinocytes 18 and cultured in a keratinocyte growth medium between time T2 and time T3 to create a tissue sample 24 including the three-dimensional dermis layer of cellular matter 20 and a three-dimensional keratinocyte epidermis layer 22. This tissue sample 24 can be placed on an insert 30 within a culture dish and exposed to an air-liquid interface between time T3 and time T4. In some cases, the process depicted in FIG. 1C is substantially the same as the process depicted in FIG.1 A except that the tissue sample 24 is subjected to radiation at a time T3’ betw een time T3 and time T4. In some embodiments, this radiation involves the sample 24 being exposed to X-ray radiation at time T3’. In some embodiments, time T3’is two days after time T3 and three days before time T4, meaning that time T4 is five days after time T3.

[0060] In some cases, the radiation applied at time T3’ can affect the three-dimensional keratinocyte epidermis layer 22 and / or the three-dimensional dermis layer of cellular matter 20 at time T4 after the tissue sample 24 is complete. For example, the radiation applied at time T3’ can induce radiation dermatitis. This can involve a thinning of the keratinocyte epidermis layer 22 depending on an intensity7and / or duration of radiation applied at time T3’. For example, more intense radiation can result in a thinner three-dimensional keratinocyte epidermis layer 22, and less intense radiation can result in a thicker three-dimensional keratinocyte epidermis layer 22. Radiation can also have an effect on the three-dimensional dermis layer of cellular matter 20 in some cases. In some cases, applying radiation at time T3’ can simulate the effects of radiation on human skin so that the tissue sample 24 reflects an accurate sample of human skin that has been exposed to radiation dermatitis.

[0061] Referring now to FIGS. 1 A-1B, the process for generating a bioprinted 3D tissue sample can include one or more steps in addition to or alternatively to the steps depicted in FIGS. 1A-1B. In some embodiments, a bioprinted human skin model replicates agespecific and disease-specific characteristics of skin based on spatially -resolved cellular senescence profiles. These profiles can be obtained from high-dimensional tissue mapping methodologies including, but not limited to, spatial transcriptomics, spatial proteomics, multiplex imaging, and single-cell profiling. Human skin samples from different age groups, anatomical locations, environmental exposures, or pathological states can be analyzed to create a molecular senescence, or biological skin aging, blueprint. TheAttorney Docket No : 07039-2350W01 / 2023-339 blueprint can define the distribution, abundance, molecular phenoty pe, and microenvironmental context of senescent cell subtypes, including senescent fibroblasts and melanocytes, and optionally senescent keratinocytes, endothelial cells, immune cells, and adnexal lineage cells. This blueprint can be generalized for defined demographic cohorts or tailored to produce customized patient-specific constructs to evaluate individualized aging, rejuvenation therapies, and regenerative outcomes.

[0062] Cellular senescence in three-dimensional bioprinted tissue samples can refer to the gradual decline in cellular function that occurs as cells experience stress before, during or after the bioprinting process, leading the cells to enter a non-dividing but metabolically active state. In bioprinted skin constructs, senescence can arise from factors such as shear stress during extrusion, suboptimal bioink composition, limited nutrient or oxygen diffusion in thick tissues, or inflammation-like biochemical cues. In addition, in bioprinted skin constructs, senescence can be pre-induced in bioinks with replicative, stress-based, or injury-based mechanisms. Senescent cells can show altered morphology, increased expression of some markers, and secretion of pro-inflammatory molecules collectively known as the senescence-associated secretory’ phenotype (SASP). In some embodiments, senescence can be induced or enriched using replicative, stress-based, or injury-based mechanisms. These mechanisms include any one or combination of DNA-damaging agents, oxidative stressors, mitochondrial stressors, telomere shortening, UV light exposure, oncogene activation, or immune-mediated pathways.

[0063] In some cases, fibroblasts of the bioink used to create the three-dimensional dermis layer of cellular matter 20 can be seeded to induce senescence. For example, the fibroblasts can be seeded at a concentration (e.g., at 10 x H cells / cm2) and the fibroblasts can be incubated for a period of time (e.g., 5 hours). In some cases, after the fibroblasts are incubated for the penod of time, the fibroblast can be added to a growth media including etoposide at a concentration (e.g., 15 pM) and maintained under standard culture conditions. The etoposide-containing media can be refreshed every 24 hours in some examples. After 48 hours, the etoposide media can be removed washed twice with PBS. In some cases, the etoposide media can be replaced with fibroblast growth media and incubated for up to 10-14 days, the fibroblast growth media refreshed every 48 hours. This can allow an emergence of senescent phenoty pes. Senescence can be be confirmed one or more biomarkers, such as SA- -gal. p!6INK4a, p21, v-112AX. SASP cytokine secretion, and ECM degradation signatures.Attorney Docket No : 07039-2350W01 / 2023-339

[0064] In some examples, epidermal melanocytes can be seeded at a concentration (e.g., 10 x 103cells / cm2) and can be incubated for at least 5 hours. After incubation, growth media including 10 pM etoposide at a concentration (e.g., 10 pM) can be added to the melanocytes and maintained cells under standard culture conditions. The etoposidecontaining growth media can be refreshed every' 24 hours. In some cases, after the growth medium has been applied for 48 hours, the etoposide media can be removed and the epidermal melanocytes can be washed twice with PBS. Melanocyte growth media can be added again and the melanocytes can be incubated for 12 days, the grow th media being changed every' other day. The melanocyte growth medium can be maintained for 10-14 days to induce senescence. The resulting product can include senescent and non-senescent cell populations incorporated into hydrogel-based bioinks prepared from natural and / or synthetic biomaterials optimized for mechanical stiffness, viscoelasticity, and biochemical signaling associated with chronological or photoaging.

[0065] The fibroblast bioink and the melanocyte bioink can be deposited in a way that retains senescence. For example, deposition of the bioink can be carried out in a layer-by-layer bioprinting process to create a multilayer dermal structure (e.g., three-dimensional dermis layer of cellular matter 20) with fibroblasts distributed according to a senescence blueprint. Senescent cell placement can be aligned to anatomical structures such as papillary vs. reticular dermis, vascular corridors, pigmentary niches, or fibrotic regions. Placement of fibroblasts can be guided by’ a patient-specific spatial map. which provides a blueprint indicating where senescent cells should be positioned within the dermal architecture, allowing the printed tissue to more accurately reflect the patient’s cellular distribution.

[0066] After printing of the three-dimensional dermis layer of cellular matter 20 is complete, the three-dimensional dermis layer of cellular matter 20 can be crosslinked using thermal, ionic, or photo-initiated methods and maintained in fibroblast media to allows ECM deposition reflective of age-related remodeling, such as elastosis or collagen fragmentation. The three-dimensional dermis layer of cellular matter 20 can be crosslinked either through incubation at 37°C or by exposure to light when a photo-initiator is added in the hydrogels. The bioprinted dermis construct can subsequently be maintained in fibroblast growth media for several days under standard culture conditions.

[0067] In some embodiments, melanocyte cells can be seeded or bioprinted as a number (e.g., 2, 3 or another number) of layers on top of the three-dimensional dermis layer of cellular matter 20 to form an initial portion of the epidermal layer. This layer can representAttorney Docket No : 07039-2350W01 / 2023-339 the three-dimensional melanocyte epidermis layer 21. In some embodiments, a melanocyte-to-keratinocyte ratio can be important for achieving physiologically relevant pigmentation and can range from 1:3 to 1:10, with a density of melanocytes (e.g., 60,000 cells per cm2or greater). Senescent melanocytes can also be incorporated according to the patient-specific spatial transcriptomics blueprint and can be printed concurrently with the melanocyte population. Following bioprinting, the constructs can be incubated in melanocyte growth medium for 3-5 days to support melanocyte proliferation and maturation.

[0068] Keratinocyte cells can be seeded or bioprinted in a number (e.g., within a range from 2-5) of layers layers on top of the melanocyte cells (three-dimensional melanocy te epidermis layer 21) to complete a formation of epidermal layer. These keratinocyte cells can represent the three-dimensional keratinocyte epidermis layer 22 comprising epidermal keratinocytes of FIG. IB. In some cases, the tissue construct is incubated in keratinocyte growth media for 2-5 days to allow formation of the epidermis layer.

[0069] In some cases, the 3D bioprinted bio-skin age construct can be transitioned to airliquid interface (ALI) using epidermal differentiation media to achieve cornification, functional tight junction formation, and stratum comeum maturation. Examples of enhancements that can be added in some embodiments include appendageal structures (e.g., hair follicles, glands), immune cell incorporation, vascularization, perfused microfluidic chip integration, and mechanical loading to simulate age-related stress conditions.&& >& >Attorney Docket No : 07039-2350W01 / 2023-339>&Table 3

[0070] In some examples, dermal bioink can be prepared by primary fibroblasts in the range of 100,000 to 500,000 cells / mL which can be homogeneously mixed with a hydrogel before bioprinting. An epidermal bioink can include within a range from 300,000 cells / cm2to 500,000 cells / cm2, or within a range from 600.000 cells / cm2to 1,000,000 cells / cm2. The epidermal bioink can be homogeneously mixed with a natural or synthetic hydrogel and melanocyte cells which are mixed with natural or synthetic hydrogel. In some cases, the melanocyte to keratinocyte ratio can be, 1:3, 1:5, 1:10, or another ratio. In some cases, the number of keratinocyte cells can be less than 60,000 cells / cm2. A dermal bioink of senescence fibroblasts and an epidermal bioink of senescence melanocytes can be prepared similarly while a number of cells incubated inside hydrogels is calculated using a patient specific spatial map.

[0071] In some embodiments, deposition of a skin bioink can be performed in a layer-by-layer, computer-controlled patterning process to form a 3D dermis layer in which dermal fibroblast cells can be suspended within a hydrated matrix or an aqueous buffer solution. The printing pattern can be defined by a spatial senescence blueprint that dictates microanatomical placement of senescent versus non-senescent fibroblasts.

[0072] In some cases, a bioprinted three-dimensional dermis layer (e.g., three-dimensional dermis layer of cellular matter 20) can be crosslinked by incubation at 37°CAttorney Docket No : 07039-2350W01 / 2023-339 for 10-90 minutes, temperature-dependent on hydrogel polymer composition. In some cases, three-dimensional dermis layer of cellular matter 20 can be crosslinked by controlled exposure to light at target wavelengths (e.g., within a range from 365 to 405 nm) for an amount of time (e.g., within a range from 15 to 120 seconds) in cases where a photoinitiator (e.g., LAP, Irgacure, riboflavin) is included in the bioink. Both thermal and photochemical crosslinking strategies can be used independently or in combination to modulate dermal stiffness and extracellular matrix (ECM) permeability to replicate age-associated matrix phenotypes, such as fibrosis, elastosis, glycation, and collagen fragmentation.

[0073] In some emboidments, following crosslinking, the three-dimensional dermis layer of cellular matter 20 can be incubated in fibroblast growth media for 3-5 days at 37°C, 5% CO?, and >95% humidity under static or perfused culture conditions, enabling fibroblast viability, ECM deposition, and senescence-associated SASP modeling.

[0074] In some embodiments, melanocyte cells can be seeded or bioprinted in 2-3 layers on top of the bioprinted dermis to form a first portion of an epidermal layer. Media composition can be modified to include melanogenic cues or UV-free pigmentation induction. The tissue construct can be incubated in melanocyte grow th media for 3-5 days to support melanocyte proliferation, dendritic extension, and pigment-transfer competence.

[0075] The melanocyte-to-keratinocyte cell ratio can be optimized to achieve physiologically relevant pigmentation levels. The melanocyte-to-keratinocyte cell ratio can be within a range from 1:3 to 1: 10, and the melanocyte density can be less than 60,000 cells / cm2to maintain pigment continuity. Senescent melanocytes can be spatially mapped to mimic pigmentary aging conditions including solar lentigines, hypopigmented patches, and perifollicular pigment decline.

[0076] In some cases, keratinocyte cells can be seeded or bioprinted in 2-5 cell layers over the melanocyte layer to complete formation of an epidermal compartment. The tissue construct can be incubated in keratinocyte growth media for 2-5 days to enable keratinocyte proliferation, stratification into basal spinous granular layers, and to establish an epidermal barrier phenotype.

[0077] In some cases, a bioprinted skin construct can be transferred to an air-liquid interface (ALI) culture, a phase that enables epidermal stratification, barrier formation, and terminal keratinocyte differentiation. The construct is incubated in skin differentiation media formulated to promote formation of the stratum granulosum and stratum corneum, tight junction assembly, and lipid barrier maturation.Attorney Docket No : 07039-2350W01 / 2023-339

[0078] In some embodiments, a-Melanocyte Stimulation Hormone (a-MSH) can be incorporated into the differentiation media at a concentration range of 50 nM to 500 nM for a period of 5 days to accelerate melanin biosynthesis and enhance pigment transfer from melanocytes to surrounding keratinocytes. a-MSH can be introduced gradually or in a stepwise dose-escalation manner to modulate pigmentation intensity and uniformity.

[0079] The differentiation media can be refreshed every 2-3 days to maintain optimal nutrient balance, remove metabolic waste, and sustain melanogenic and differentiation signals. During this period, the media composition can be optionally be supplemented with additional pigmentation-modulating or rejuvenation-enhancing agents such as tyrosinase regulators, cyclic AMP agonists, senolytic compounds, senomorphic compounds, or antioxidants, without deviating from the core inventive method.

[0080] H&E-stained bioprinted pigmented skin samples can demonstrate a well-organized, spatially stratified epidermis consisting of basal, spinous, granular, and cornified layers, along with a structurally intact and cellularly viable dermal layer across all a-MSH-treated groups. In some cases, epidermal thickness exhibits dose-responsive modulation, where higher a-MSH concentrations correlate with improved stratification and more homogeneous keratinocyte layer organization.

[0081] In some cases, pigment deposition can appear enhanced without evidence of tissue dysmorphia, disruption of dermal-epidermal j unction integrity, excessive keratinocyte proliferation, or necrotic change. Collectively, these data confirm that a-MSH exposure during the ALI phase can effectively supports epidermal integrity, induces physiologically relevant pigmentation, and can promote regenerative activity' or age-related phenoty pe correction while maintaining native-like tissue morphology' and biomechanical structure.Attorney Docket No : 07039-2350W01 / 2023-339 >Table 4

[0082] In some embodiments, melanocytes included within the epidermal bioink can undergo controlled pigment induction, enabling a construct to recapitulate physiologic melanogenesis and pigment transfer processes. By embedding melanocytes directly within printed epidermal layers at controlled physiological ratios, the model can replicate functional pigmentation, including melanin synthesis and intracellular storage, melanocyte dendrite extension, melanosome transport and transfer to keratinocytes, and pigment patterning reflective of aging or disease state. This ab i 1 i ty to intentionally induce and modulate pigmentation within a fully stratified bioprinted human skin construct can represent a unique and defining aspect of skin modeling technology, enabling direct modeling of age-related pigmentary disorders, photodamage profiles, racial / ethnic-specific melanin biology, and therapeutic pigmentation modulation in a laboratory -grown human skin system.

[0083] A maintenance media can be selected to sustain the physiological integrity and long-term viability of the bioprinted skin construct. In some cases, drug screening or therapeutic testing can begin after keratinocyte cells have fully differentiated to form a stratified, functionally mature epidermis with a competent barrier.

[0084] In some cases, maintenance media can play an important role in preserving epidermal homeostasis, supporting mitochondrial and metabolic activity, sustaining nutrient exchange, and preventing structural deterioration of both the epidermal and dermal compartments. This can include prevention of basement membrane disruption, dermal fibroblast apoptosis, and pigment dilution at the melanocyte-keratinocyte interface.

[0085] Some bioprinted skin studies can rely on continued exposure to differentiation media as the final culture stage. However, extended incubation in high-calcium, differentiation-inducing formulations can lead to excessive terminal differentiation, hypercomification, diminished proliferative capacity, and premature deterioration of tissueAttorney Docket No : 07039-2350W01 / 2023-339 barrier properties. Hypercomification can adversely affect tight junction organization, melanocyte dendricity, paracrine signaling, and transepithelial electrical resistance , thereby compromising the physiological relevance of the model for screening applications.

[0086] A transition to maintenance media following epidermal maturation can stabilize an epidermal barrier and prevent pathological cornification, while preserving functional melanocyte activity, controlled pigmentation phenoty pes, and senescence-associated biological pathways. This shift can enable extended culture duration and maintain biomimetic function throughout testing.

[0087] Following maturation of the epidermal layer during the air-liquid interface (ALI) phase, bioprinted constructs can be transferred to maintenance media for approximately 10-14 days. This approach can allow the bioprinted constructs to remain fully viable and test-ready for drug-screening, cosmetic evaluation, and mechanistic profiling for up to approximately 24 days from the start of ALI culture.Table 5

[0088] In some cases, the model can incorporate new culture methodologies, maintenance media systems, and viable pigmentation-capable epidermal compartments, enabling significantly extended culture duration and sustained tissue integrity. This enhanced stability can support long-term evaluation of therapeutics for up to 24 days or longer, allowing dosing regimens, exposure cycles, and endpoint analyses that are aligned with FDA preclinical expectations.

[0089] Deposition of bioink can be performed in a layer-by-layer manner to form a 3D dermis layer in which dermal fibroblast cells are suspended in a hydrated hydrogel-based matrix. Dermal bioink can be prepared by primary fibroblasts in the range of 100,000 to 500,000 cells / mL which homogeneously mixed with the hydrogel before 3D bioprinting. In some embodiments, The epidermal bioink can be composed of 300.000 to 500,000 cells / cm2, or 600,000 to 1,000,000 cells / mL, homogeneously mixed with a natural or synthetic hydrogel.Attorney Docket No : 07039-2350W01 / 2023-339

[0090] In some embodiments, the bioprinted 3D dermis layer can be crosslinked either by incubation at 37°C for 10-90 minutes, tailored to hydrogel chemistry’, or by photocrosslinking at specific wavelengths (e.g., 365-405 nm) for 15-120 seconds when a photoinitiator is included. Following stabilization, the dermal construct can be incubated in fibroblast growth media for 3-5 days at 37°C and 5% CO2 to promote extracellular matrix deposition, intercellular communication, and viability maintenance.

[0091] Keratinocyte cells can be seeded or bioprinted in 2-5 layers atop the dermis to form the epidermal compartment, in some cases. The construct can be incubated in keratinocyte grow th media for 2-5 days to allow stratification and junctional organization prior to disease induction.

[0092] In the next step, the bioprinted skin construct can be transferred to an air-liquid interface (ALI) culture and incubated in skin differentiation mediate achieve barrier maturation. Human interleukin-4 (IL-4) at a concentration of 20 nM can be added to the differentiation media for 48 hours to induce an atopic dermatitis-like inflammatory’ phenotype. Media can be refreshed every 2-3 days to maintain active immune stimulation and preserve epidermal structural integrity during disease modeling.

[0093] In some cases, this controlled inflammatory induction allows reproducible onset of clinical hallmarks (e.g., dysregulated differentiation, altered K14 / involucrin distribution, barrier impairment), without compromising long-term viability.

[0094] Referring now to FIG. 2, a three-dimensional printer can print a set of dermis layers printed by a 3D bioprinter. To create these samples, the 3D bioprinter can deposit layers of a bioink in a layer-by-layer manner until the three-dimensional dermis layers of cellular matter are formed. These samples can be placed into a fibroblast growth media as discussed above. Referring now to FIGS. 3A-3K. several different designs of insert can be used in the process to create tissue samples. Each of these designs allows the sample to rest on the insert elevated above a floor of a culture dish. Liquid can fill the culture dish up to a predetermined level such that part of the sample is exposed to media and part of the sample is exposed to air. FIG. 4 illustrates a process for creating polyacrylamide hydrogels.

[0095] Referring now to FIGS. 5A-5D, a patient-specific map can be used to deposit bioink in a layer-by-layer manner according to a senescence biological aging model. The patient-specific map can be used to deposit dermal fibroblasts, epidermal keratinocytes, epidermal melanocytes, or any combination thereof. FIG. 5A depicts a map of single-cell RNA sequencing (scRNA-seq) of face skin cells of a population that has been exposed toAttorney Docket No : 07039-2350W01 / 2023-339 radiation (e.g., sunlight). FIG. 2B depicts a map of visium spatial transcriptomics of sun-exposed human skin, including a prevalence of the CDKN1A and the CDKN2A genes. The CDKN1A and the CDKN2A genes can correspond to skin cells associated with high risk for melanoma and other kinds of cancer. FIG. 5C indicates a map of clusters in visium spatial transcriptomics including highly concentrated CDKNIA-expressing spots in clusters. These maps can be beneficial for creating the senescence biological aging model, because areas that are highly concentrated for CDKN1A and CDKN2A can indicate where senescent cells should be positioned within the three-dimensional dermis layer of cellular matter 20 to create an accurate sample of human tissue. FIG. 5D depicts a plot diagram indicating a proportion of highly-CDKNIA (P21)-expressing spots with UV exposure and chronological age. This confirms that higher proportion of CDKN1A indicates more senescent cells.

[0096] FIG. 6 is a flow diagram illustrating how a print head 10 and a seeding device 18 can deposit material to fabricate a three-dimensional sample of human tissue. For example, a print head 10 can deposit a bioink in a layer-by-layer manner to create a three-dimensional dermis layer 20 of cellular matter, wherein the bioink comprises dermal fibroblasts suspended in an aqueous buffer solution (102). In some examples, creating the bioink involves mixing the dermal fibroblasts and monomer molecules in an aqueous buffer solution. In some examples, mixing the dermal fibroblasts and monomer molecules comprises causing a concentration of the dermal fibroblasts in the aqueous buffer solution to be within a range from 2 x 104- milhlceetlelrs(r—mL) - to 40 x 106mL ' and causing a concentration of the monomer molecules to be within a range from 2t40 The monomer molecules can comprise collagen methacrylamid. The aqueous buffer solution can also include sodium hydroxide (NaOH) and phosphate-buffered saline in some examples. The three-dimensional dimensional dermis layer 20 of cellular matter can be exposed to a fibroblast growth medium for a first period of time in some cases (104).

[0097] After the first period of time, the three-dimensional dermis layer 20 of cellular matter can be seeded with epidermal karatinocytes (106) using a seeding device 18. In some cases, seeding the three-dimensional dermis layer of cellular matter 20 comprises placing the epidermal keratinocytes on a surface of the three-dimensional dermis layer of cellular matter according to a predetermined concentration. In some examples, theAttorney Docket No : 07039-2350W01 / 2023-339predetermined concentration is within a range from 50,000 - ce?itimeter(c?n) - tocells cells 400,000 — ar - In some examples, the predetermined concentration is 300,000 — err --

[0098] The seeded three-dimensional dermis layer of cellular matter 20 can be exposed to a keratinocyte growth medium for a second period of time (108). Adter the second period of time, the seeded three-dimensional dermis layer of cellular matter 20 can be mounted on an insert until the seeded epidermal karatinocytes form a three-dimensional keratinocyte epidermis layer 22 of cellular matter on top of the three-dimensional dermis layer of cellular matter 20 (110).

[0099] While this specification contains many specific implementation details, these should not be construed as limitations on the scope of any invention or of what may be claimed, but rather as descriptions of features that may be specific to particular embodiments of particular inventions. Certain features that are described in this specification in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable sub combination. Moreover, although features may be described herein as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a sub combination or variation of a sub combination.

[0100] Similarly, while operations are depicted in the drawings in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed, to achieve desirable results. In certain circumstances, multitasking and parallel processing may be advantageous. Moreover, the separation of various system modules and components in the embodiments described herein should not be understood as requiring such separation in all embodiments, and it should be understood that the described program components and systems can generally be integrated together in a single product or packaged into multiple products.

[0101] Particular embodiments of the subject matter have been described. Other embodiments are within the scope of the following claims. For example, the actions recited in the claims can be performed in a different order and still achieve desirable results. As one example, the processes depicted in the accompanying figures do not necessarilyAttorney Docket No : 07039-2350W01 / 2023-339 require the particular order shown, or sequential order, to achieve desirable results. In certain implementations, multitasking and parallel processing may be advantageous.

Claims

Attorney Docket No : 07039-2350W01 / 2023-339CLAIMSWhat is claimed is:

1. A method comprising:depositing a bioink in a layer-by-layer manner to create a three-dimensional dermis layer of cellular matter, wherein the bioink comprises dermal fibroblasts suspended in an aqueous buffer solution;exposing the three-dimensional dermis layer of cellular matter to a fibroblast grow th medium for a first period of time;after the first period of time, seeding the three-dimensional dermis layer of cellular matter with epidermal keratinocytes;exposing the seeded three-dimensional dermis layer of cellular matter to a keratinocyte growth medium for a second period of time; andafter the second period of time, mounting the seeded three-dimensional dermis layer of cellular matter on an insert until the seeded epidermal keratinocytes form a three-dimensional epidermis layer of cellular matter on top of the three-dimensional dermis layer of cellular matter.

2. The method of claim 1. further comprising creating the bioink by mixing the dermal fibroblasts and monomer molecules in an aqueous buffer solution.

3. The method of claim 2, wherein mixing the dermal fibroblasts and monomer molecules comprises causing a concentration of the dermal fibroblasts in the aqueousbuffer solution to be within a range from 2 X 104- millileter - (mL) to 40 X 106mL and causing a concentration of the monomer molecules to be within a range from 2 milligrams (mg)r. mg- mL tO 4(J - mL .

4. The method of claim 2, w herein the monomer molecules comprise collagen methacrylamide, and wherein the aqueous buffer solution further comprises sodium hydroxide (NaOH), and phosphate-buffered saline.

5. The method of claim 1, wherein seeding the three-dimensional dermis layer ofAttorney Docket No : 07039-2350W01 / 2023-339 cellular matter comprises placing the epidermal keratinocytes on a surface of the three-dimensional dermis layer of cellular matter according to a predetermined concentration.

6. The method of claim 5, wherein the predetermined concentration is within a range from 50,0007. The method of claim 5. wherein the predetermined concentration is300,000 cnr8. The method of claim 1, wherein the first period of time comprises 5 days, and wherein the second period of time comprises 2 days.

9. The method of claim 1. further comprising culturing the seeded three-dimensional dermis layer of cellular matter in a keratinocyte differentiation media when the seeded three-dimensional dermis layer of cellular matter is mounted on the insert so that a first portion of the seeded three-dimensional dermis layer of cellular matter is exposed to air and a second portion of the seeded three-dimensional dermis layer of cellular matter is exposed to the keratinocyte differentiation media.

10. The method of claim 9, w herein the insert is permeable such that a surface of the insert defines one or more openings so that the keratinocyte differentiation media can pass through the one or more openings.

11. The method of claim 1, further comprising:creating a set of polyacrylamide hydrogels, each polyacrylamide hydrogel of the set of polyacrylamide hydrogels having an elastic modulus;identifying, based on the elastic modulus of each polyacrylamide hydrogel of the set of polyacrylamide hydrogels, an elastic modulus at which biological functions of skin cells are highest; anddepositing the bioink according to the elastic modulus.

12. The method of claim 1, wherein depositing the bioink in the layer-by-layer manner comprises:Attorney Docket No : 07039-2350W01 / 2023-339 creating a senescence biological aging model for depositing the bioink in the layer-by-layer manner, the senescence biological aging model being indicating locations where senescent cells should be positioned within the three-dimensional dermis layer of cellular matter; anddepositing the bioink according to the senescence biological aging model based on patient-specific spatial mapping.

13. The method of claim 1, wherein the bioink comprises a dermal bioink, and wherein the method further comprises:after the first period of time, depositing a second bioink in a layer-by-layer manner on top of the three-dimensional dermis layer of cellular matter to create a base epidermis layer of cellular matter, wherein the second bioink comprises epidermal menalocytes, wherein seeding the three-dimensional dermis layer of cellular matter with epidermal keratinocytes further comprises seeding the base epidermis layer of cellular matter, and wherein the seeded epidermal keratinocytes and the base epidermis layer form the three-dimensional epidermis layer of cellular matter on top of the three-dimensional dermis layer of cellular matter.

14. The method of claim 13, further comprising crosslinking the three-dimensional dermis layer of cellular matter by:incubating the three-dimensional dermis layer of cellular matter at a temperature for an amount of time; orexposing the three-dimensional dermis layer of cellular matter to light having wavelengths within a range from 280 nm to 405 nm.

15. The method of claim 13, further comprising exposing the base epidermis layer of cellular matter to a melanocyte grow th medium.

16. The method of claim 13, wherein a melanocyte-to-keratinocyte ratio in the three-dimensional epidermis layer of cellular matter is within a range from 1:3 to 1:10.

17. The method of claim 13, further comprising placing an air-to-liquid interface into the insert with the seeded three-dimensional mass of cellular matter.

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