Biological scaffolds, products containing biological scaffolds, and methods of using the same

Abstract

The present disclosure provides biological scaffolds, methods for their synthesis, and methods for their use. The biological scaffolds include at least two components: mammalian cell platelet lysate and adipose tissue-derived cell fraction (ATDCF) cells. Formulations containing the biological scaffold(s) can be gel, semi-solid, or solid substrates, at least one surface of which contacts the biological scaffold to form a biocompatible material. The biological scaffolds and biological scaffold-containing formulations can find utility in a wide range of medical device technologies and can be used for therapeutic purposes or to conduct experiments testing various drugs in vitro and in vivo. [Selected Figure] Figure 1

Description

[Technology Field]

[0001] Cross-reference of related applications This international PCT application claims priority and benefits of U.S. Provisional Patent Application No. 62 / 457,366, filed on 10 February 2017, and the entire contents of that application are incorporated herein by reference as forming part of this specification.

[0002] Statements concerning research funded by the federal government This invention was completed with the support of the United States Government under Federal Grant No. R21DK094254 from the National Institutes of Health. The United States Government has certain rights to this invention. This invention was completed using parts of the facilities of the Cell Biology and Bioimaging Core, operated by the COBRE (NIH 8 P20-GM103528) and NORC (NIH 2P30-DK072476) Centers, which are partially funded by grants from the National Institutes of Health.

[0003] This disclosure relates to novel biological scaffolds derived from adipose tissue, products utilizing such scaffolds, and methods of using them. [Background technology]

[0004] While isolated human pre-adipocytes and adipocytes are widely used in two-dimensional (2D) cultures to identify signaling pathways associated with healthy and diseased adipose tissue, standard 2D culture models are substantially limited. Two-dimensional models cannot replicate the progression of adipocyte hyperplasia and hypertrophy characteristic of clinical obesity. Due to the biomechanical constraints of 2D models, it is impossible to create the classic unilocular or "signet ring" adipocytes characteristic of human obesity. These distinctive structures are only possible in three-dimensional (3D) models. Unlike 2D models, 3D human SVF cell constructs created using tissue engineering combine biochemical, biomechanical, and biophysical signals that influence gene expression, cell polarity, and intercellular interactions in a biomimetic environment. In contrast to 3D models, 2D monolayer models cannot capture circulating hematopoietic cells residing in natural adipose tissue under both physiological and pathological conditions. [Overview of the project] [Means for solving the problem]

[0005] The specific novel features of the present invention described below are described in the claims appended to this specification, but those skilled in the art will understand that various omissions, modifications, substitutions, and changes are possible in the exemplary forms and details of the present invention and its operation without departing from the spirit of this disclosure, and therefore the present invention is not intended to be limited to those described in detail. No feature of this disclosure is essential or indispensable unless it is explicitly stated to be so.

[0006] The present disclosure provides a functional, humanized, cell biological scaffold fabricated in tissue engineering, which enables the production of various biocompatible products for regenerative medicine, tissue remodeling, and experimental models for the study of cell growth and differentiation, as well as other drug discovery applications. In some embodiments, the biological scaffold comprises at least two components, namely mammalian platelet lysate and an adipose tissue-derived cell fraction (ATDCF). In related embodiments, the biological scaffold comprises, but is not limited to, one or more of stromal vascular fraction cells, adipose-derived stromal cells, adipose-derived stem cells, bone marrow-derived mesenchymal stromal cells, and bone marrow-derived mesenchymal stem cells.

[0007] In various embodiments, the biological scaffolds exemplified herein have numerous applications for the study of cell differentiation and the generation of new tissues, such as fat, cartilage, bone, blood cells, blood vessels, and combinations thereof. Further, the biological scaffolds can be seeded with any cell type that requires a physiological environment close to the natural environment to mimic disease processes. Specific exemplary embodiments can include the generation of patient-derived xenograft tumors or other tumors from standard cancer cell lines, which can be cultured in two-dimensional or three-dimensional constructs in tissue culture or, for example, transplanted in vivo in a mouse model.

[0008] In further embodiments, the biological constructs described herein can be incorporated into further utilizable products.

[0009] In some embodiments, an exemplary apparatus incorporating the scaffolds disclosed herein may include a) one or more cell components derived from an adipose tissue-derived cell fraction (ATDCF), mammalian platelet lysate, and optionally, a cell population, and b) a combination with a human-derived and / or silk-based static three-dimensional (3D) culture having a microfluidic morphology with an inlet and an outlet. The combination of these cells and the biological scaffold enables short-term and long-term 3D in vitro culture of various combinations of one or more cell subtypes present in the ATDCF, or in vivo transplantation into both small and large animal models. In certain embodiments, the present disclosure provides a humanized, adipose depot engineered in tissue engineering, comprising a combination of an adipose tissue resident stromal vascular fraction (SVF) cell population and mammalian platelet lysate, wherein the platelet lysate can be a human platelet lysate. This cell / biological scaffold combination enables short-term and long-term three-dimensional in vitro culture of various combinations of one or more cell subtypes present in the SVF, or in vivo transplantation into both small and large animal models. Silk, human platelet lysate, and ATDCF (SVF cells) enable strong cell adhesion, adipocyte differentiation, and maintenance of cell diversity associated with both healthy and metabolically diseased human adipose depots. These constructs can be cultured for over several weeks while maintaining their macroscopic and cellular structure, as well as their function. Additionally, the biological scaffolds proposed herein are bioactive, biocompatible, do not contain donor DNA, are of human origin, are suitable for both autologous and allogeneic transplantation, and support stromal / stem cell proliferation.

[0010] Both static and microfluidic in vitro constructs can be cultured for short-term or long-term periods while maintaining their macroscopic and cellular structures, as well as their functions. In addition, the biological scaffolds proposed herein are bioactive, biocompatible, do not contain donor DNA, are of human origin, are suitable for both autologous and allogeneic transplantation, and support stromal / stem cell proliferation.

[0011] In accordance with this discovery, the objective of this disclosure is to provide a static microfluidic 3D in vitro platform for adipose tissue-derived stem / stromal cells, stromal vascular fraction cells, or other primary cells present within adipose tissue.

[0012] A further objective of this disclosure is to provide adipose tissue depots derived from mice or other small or large animals in vitro from humanized mice.

[0013] A further objective of this disclosure is to provide methods for pharmacological drug testing and for the study of the tumor microenvironment.

[0014] Other purposes and benefits of this disclosure are apparent from the following description.

[0015] The following drawings constitute part of this specification and are included to further demonstrate specific aspects of this disclosure. This disclosure can be better understood by referring to one or more of these drawings in combination with the description of the specific embodiments presented herein. [Brief explanation of the drawing]

[0016] [Figure 1] The characteristics of the cells used in this study are shown below. a) Pie chart of subpopulations of GFP-Tg SVF cells used for serial SVF transplantation. 1b) Selection and concentration of CD146-CD29+ and CD146-CD34+ subpopulations cultured and grown to P2 ASC for serial GFP-Tg ASC transplantation. lc) Immunophenotypes of GFP-Tg SVF cells and GFP-Tg ASCs cultured and grown to passage 2 (P2). All experiments were repeated three times. Sample size was n=3 / replica. Numerical values ​​are reported as mean + standard deviation (μ + SD). * p<0.05, ** p<0.01, *** p<0.001. [Figure 2] This report compares GFP-Tg ASCs rich in CD29 and CD34. 2a) Immunophenotypes of CD146-CD29+ and CD146-CD34+ were selected to form GFP-Tg ASC subpopulations. Immunophenotype was based on the expression of CD29, CD31, CD34, CD45, CD11B, Sca-1, and CD105 surface antigens. 2c) Lipidogenesis differentiation of unfractionated GFP-Tg ASCs, CD146-CD29+GFP-Tg ASCs, and CD146-CD34+GFP-Tg ASCs, and 2d) Colony formation assays. All experiments were repeated three times. Sample size was n=3 / replica. Numerical values ​​are reported as mean + standard deviation (μ+SD). * p<0.05, ** p<0.01, *** p < 0.001. [Figure 3] Shows the weekly status of tissue-engineered fat inside and around GFP-Tg SVF cell grafts in mice. 3a). The scaffolds were transplanted without cells or with 500k GFP-Tg SVF cells into silk scaffolds. The scaffolds were removed 1 week, 2 weeks, or 6 weeks after transplantation. 3b). Percent hemoglobin saturation was measured immediately after transplantation and 1 week and 6 weeks after the first transplantation. 3c). Quantification of engraftment of the removed scaffolds (explants) via ImageJ analysis of the microscopic images in 3a, with sample size n = 6 / group. 3d). Scaffold mass measurements 1 week, 2 weeks, and 6 weeks after transplantation. Sample size is n = 20. Quantification correlates with microscopic images showing SVF-mediated engraftment promotion 1 week after transplantation. 3e). Weekly quantification of SVF cells correlates with persistence, proliferation, and differentiation of SVF cells up to 2 weeks. Values are reported as mean + standard deviation (μ+SD). * p < 0.05, ** p < 0.01, *** p < 0.001. [Figure 4-1] This study shows the detection of tissue-engineered adipose-derived GFP-Tg SVF cells using silk scaffold grafts from mice that underwent two consecutive transplants. Microscopic images taken 1, 4, and 6 weeks after in vivo transplantation of GFP-Tg SVF cells demonstrate persistence, proliferation, and the ability to form GFP-Tg fat depots. 4a) Confocal microscopy images and composite images of GFP, BODIPY, and DAPI in GFP-Tg SVF cell (T0) grafts during the first 6 weeks. 4b) Composite images of GFP / BODIPY / DAPI from first-sequence (T1) and second-sequence (T2) transplants of GFP-Tg SVF cells extracted after 6 weeks. Sample size: n=5. 4c) Flow cytometry analysis of GFP expression in adipose scaffolds extracted after T0, T1, and T2 transplantation. Sample size: n=18. 4d) ImageJ analysis of weekly confocal images of T0 explants. 4e) Quantitative analysis of weekly flow cytometry of GFP expression in isolated T0 GFP-Tg SVF cell grafts. 4f) GFP DNA expression in samples derived from 6-week isolated T0, T1, and T2 grafts. GFP expression was reported as an expression multiplier and normalized against GAPDH expression. 4g) Immunophenotyping based on GFP, CD29, CD31, CD34, CD45, and Sca-1 antigen expression in SVF cells isolated from 6-week T2 grafts. Numerical values ​​were reported as mean + standard deviation (μ + SD). Each group was compared to a GFP+ control. * p<0.05, ** p<0.01, *** p<0.001. Seeded GFP-SVF T2 was compared to an unseeded control group. And p<0.05. [Figure 4-2] Continuation of Figure 4-1. [Figure 5] This shows adipogenesis and functionality of GFP-Tg ASCs seeded on serial silk grafts. 5a) Microscopic images of scaffolds with (a) 500k unsorted GFP-Tg ASCs, (b) CD146-CD29+GFP-Tg ASCs, or (c) CD146-CD34+GFP-Tg ASCs transplanted onto silk scaffolds, either cell-free (control) or silk scaffolds. Scaffolds were removed 6 weeks after transplantation. 5b) Images of scaffolds removed from cohorts (a) to (c). 5c) Percent hemoglobin saturation was measured in groups (a) to (c) 6 weeks after the first (T0) transplantation. 5d) Quantification of engraftment of removed scaffolds (explants) after ImageJ analysis of microscopic images in 4b, with a sample size of n=6 / group. 5e) Mass measurement of grafts from cohorts (a)–(c) after 6 weeks, and of control scaffolds (grafts without cell seeding) 5f). Quantification of SVF collected from grafts after 6 weeks, surrounding fat from cohorts (a)–(c), and the unseeded control group. The sample size was n=18 / group. Functionality was measured using 6-week T1 graft constructs by 5g) glucose uptake and 5f) glycerol secretion assay. Data were analyzed using Graphpad Prism. Two-way ANOVA was performed. Data were reported as mean + SD. * p<0.05, ** p<0.01, *** p<0.001. This is compared to the group rich in CD34. p<0.05. [Figure 6] This shows the detection of tissue-engineered fat-derived CD146-CD29+GFP-Tg ASCs and CD146-CD34+GFP-Tg ASCs using silk scaffold grafts of HFIP for 6 weeks in mice that underwent two consecutive transplants. Microscopic images of GFP-Tg CD29-rich and CD34-rich cohort (a)-(c) cells were transplanted in vivo, and after 6 weeks, demonstrated persistence, proliferation, and the ability to form GFP-Tg fat depots, similar to the unselected GFP-Tg SVF cohort. 6a) Confocal microscopic images and composite images of GFP, BODIPY, and DAPI in 6-week GFP-Tg ASC consecutive (T1) grafts. 6b) Confocal images of 6-week T1 groups rich in CD29 and CD34 reflected the observation of microangiogenesis more in the CD34-rich group than in the CD29-rich group. n=5 / group / replication. 6c) Flow cytometry analysis of GFP expression in adipose scaffolds extracted from cohorts (a)-(c) after T0 and 6d) T1 transplantation. 6e) GFP DNA expression in samples derived from extracted 6-week T0 and T1 grafts. GFP expression is reported as expression multipliers and normalized against GAPDH expression. 6f) Quantification of %GFP positivity 6 weeks after transplantation of T0 and 6g) T1 GFP-Tg ASC cell grafts. Data are reported as mean + SD. Each group is compared to a group seeded with unselected cells. * p<0.05, ** p<0.01, *** p<0.001. This is compared to the group rich in CD34. p<0.05. [Figure 7] This study demonstrates that GFP-Tg SVF cells, GFP-Tg ASCs rich in CD29, and GFP-Tg ASCs rich in CD34 infiltrate surrounding tissues to produce functional GFP-Tg fat. Confocal microscopy images of fat within 2 mm surrounding SVF and ASC constructs showed GFP positivity. 7a) Synthetic images of negative control fat and positive control fat stained with BODIPY and DAPI, respectively, in non-GFP-Tg and GFP-Tg mice. 7b) Images of fat surrounding constructs seeded with unsorted GFP-Tg SVF cells, unsorted GFP-Tg ASCs, and GFP-Tg ASCs rich in CD146-CD29 and CD34 for 6 weeks. n=5 / group / replication. 7c) Quantification of confocal images from 7a and 7b. Functionality was reported by 7d) glycerol secretion and 7e) glucose uptake assay using the T1 transplanted construct after 6 weeks. Data were reported as mean + SD. * p<0.05, ** p<0.01, *** p<0.001. [Figure 8] This document outlines the overall process involved in the development of silk-based 3D cultures. SVF cells were isolated from the inguinal white adipose tissue of GFP-Tg C57Bl / 6 mice according to an established protocol (see Materials and Methods). CD146-GFP-Tg SVF cells were sorted and selected based on CD29+ and CD34+ expression. Cells rich in CD29+, cells rich in CD34+, and unsorted cells were seeded onto silk scaffolds and transplanted into non-GFP-Tg mice, or ASC-like populations were cultured and grown up to passage 2, seeded onto silk scaffolds, and transplanted into non-Tg mice. Six weeks after transplantation, the cultured tissue constructs were removed and digested using a collagenase tissue digestion protocol (see Materials and Methods). Isolated cells were plated, grown, and transplanted sequentially. [Figure 9] This shows in vitro seeding of GFP-Tg ASCs into silk scaffolds for transplantation studies. 9a) Confocal images at 100x magnification of DAPI and GFP expression. 9b) Bright-field and synthetic images of DAPI / GFP expression in metabolically active GFP-Tg ASCs 18 hours after in vitro seeding on silk scaffolds. 9c) Confocal microscopy images of lipid-producing differentiated GFP-Tg ASCs in lipid-producing medium for 5 days. 9d) Cryo-scanning electron microscopy (cryo-SEM) images of uninoculated scaffolds at 80x magnification. 9e) Metabolic active GFP-Tg ASCs present by Alamar Blue staining and 9f) adhered by DAPI staining 2 hours and 18 hours after in vitro introduction into silk scaffolds. Relative fluorescence intensity (RFU), determined by Alamar Blue analysis, was normalized to the wet weight of the scaffold and used as a measure of cell adhesion level 2 hours and 18 hours after seeding ASCs within each scaffold group. All experiments were repeated three times. The sample size was n=5 / replica. Numerical values ​​were reported as mean + standard deviation (μ + SD). ** p<0.01. [Figure 10] This diagram shows a schematic of the process for constructing a thermoresponsive 3D culture of human stromal vascular fraction and human platelet lysate (SVF / Hapley 3D construct). Human SVF cells are isolated from digested human adipose tissue. The hSVF cells are mixed with stromal medium supplemented with an optimal concentration of hPL and incubated at 37°C while injecting 5% CO2. The thermoresponsive self-assembling matrix is ​​formed immediately upon mixing with the cell-hPL / medium combination. [Figure 11] This study demonstrates the proliferative capacity of human SVF cells cultured under 3D conditions in hPL compared to traditional 2D monolayer culture. Cell proliferation was measured via Cell Titer-Blue reagent after 3, 5, and 7 days of culture under 2D or 3D conditions. Metabolically active SVF present and adhering was quantified in vitro after introduction into silk scaffolds at 24, 48, and 72 hours. Cell counts determined by Cell Titer-Blue absorbance analysis were normalized against a cell count titration standard curve and used as a measure of cell proliferation within each hPL matrix group. All experiments were repeated three times. Sample size was n=3 / replica. Numerical values ​​are reported as mean + standard deviation (μ+SD). * p<0.05. [Figure 12] This study demonstrates hSVF spheroid formation in 3D hPL culture. Microscopic images show that spheroid formation occurs at low hPL concentrations. Both spheroid size and density directly correlate with the percentage hPL concentration in the interstitial medium. All experiments were repeated three times. Sample size was n=3 / replica. Numerical values ​​are reported as mean + standard deviation (μ + SD). * p<0.05. [Figure 13] This study demonstrates the correlation between cell number and cell-mediated matrix gelation and matrix stability. All experiments were repeated three times. Sample size was n=5 / replica. Numerical values ​​are reported as mean + standard deviation (μ+SD). * p<0.05. [Figure 14] This study describes the proliferation and spheroid formation behavior of the stromal vascular fraction when cultured with an hPL-based matrix. Cell proliferation was measured via Cell Titer-Blue reagent after 3, 5, and 7 days of culture under 2D or 3D culture conditions. Metabolically active SVF present and adhering to the matrix was quantified 24, 48, and 72 hours after inoculation. Cell counts determined by Cell Titer-Blue absorbance analysis were normalized to a cell count titration standard curve and used as a scale for cell proliferation within each hPL matrix group. Cell proliferation and spheroid formation per field of view (FOV) were determined by phase contrast or confocal laser microscopy after staining with Hoescht (nucleus) or Bodipy (lipid vacuole). Both spheroid size and density directly correlated with the percentage hPL concentration in the stromal medium. Microscopic images demonstrate that spheroid formation occurs at low hPL concentrations. All experiments were repeated three times. The sample size was n=5 / replica. Numerical values ​​were reported as mean ± standard deviation (μ±SD). * p<0.05. [Figure 15] Microscopic images of unseeded and human SVF cells seeded on human platelet lysate scaffolds are shown. Confocal images stained with Hoescht and Bodipy dyes were synthesized with 3D human platelet lysate cultures maintained in vitro for 3 weeks, either with or without lipidogenesis differentiation medium. The same cultures were fixed for cryo-scanning electron microscopy (cryo-SEM) and imaged at a magnification of 30–50 μm. All experiments were repeated three times. Sample size was n=3 / replica. [Figure 16] This study demonstrates the in vitro formation of human white adipose tissue (hWAT) and beige / bright adipose tissue (hbAT) using SVF cells and an hPL-based matrix. SVF cells were seeded in an hPL matrix, and lipid synthesis was induced for two weeks using white or beige / bright lipid synthesis cocktails. Confocal images (100x magnification) stained with Hoescht and Bodipy dyes were synthesized with 3D human platelet lysate cultures. All experiments were repeated three times. Sample size: n=3 / replica. [Figure 17] This study quantifies human white adipose tissue (hWAT) and beige / bright adipose tissue (hbAT) formed in culture using SVF cells and an hPL-based matrix. SVF cells were seeded in an hPL matrix, and lipid synthesis was induced for two weeks using white or beige / bright lipid synthesis cocktails. Confocal images (100x magnification) of the SVF / hPL matrix constructs and positive control human adipose tissue were stained with Hoesch and Bodipy dyes. Confocal images were quantified using Cell Profiler software (www.cellprofiler.org). All experiments were repeated three times. Sample size was n=5 / replica. Numerical values ​​are reported as mean ± standard deviation (μ±SD). * p<0.05. [Figure 18] This shows in vivo grafts of hSVF cell / human platelet lysate bioscaffolds in nude mice. Subcutaneous grafts were harvested after 8 weeks, fixed with 4% paraformaldehyde, and sectioned into 10 μm slices. These sections were placed on imaging slides, stained with the nucleus (hematoxylin) or neutral lipids (eosin), and visualized with a slide scanner. All experiments were repeated three times. Sample size was n=3 / replica. [Modes for carrying out the invention]

[0017] A detailed description of one or more preferred embodiments is provided herein. However, it should be understood that this disclosure embodies a variety of forms. Accordingly, the specific details disclosed herein should not be interpreted restrictively, but rather as a basis for the claims and as a representative basis for instructing those skilled in the art to utilize this disclosure in any appropriate way.

[0018] Whenever the phrases "for example," "such as," or "including" are used in this specification, they are followed by the phrase "and without limitation" unless otherwise specified. Similarly, "an example" and "exemplay" should be understood as non-limiting.

[0019] The term "substantially" allows for deviations from the description as long as they do not negatively affect the intended purpose. Even if the word "substantially" is not explicitly written, descriptive terms should be understood as being modified by the term "substantially." Therefore, for example, the phrase "the lever extends vertically" means "the lever extends substantially vertically" unless the lever requires a precise vertical orientation to perform its function.

[0020] The terms “comprising,” “including,” “having,” and “involving” (as well as “comprises,” “includes,” “has,” and “involves”) are used interchangeably and have the same meaning. Specifically, each of these terms is defined to correspond to the general definition of “comprising” in U.S. patent law, and is therefore interpreted to mean “at least the following,” and not to exclude additional features, limitations, or aspects. For example, “a process comprising steps a, b, and c” means that the process comprises at least steps a, b, and c. Wherever the term “a” or “an” is used, it is understood to mean “one or more,” unless its interpretation is meaningless in context.

[0021] The term "depot" generally refers to a specific area of ​​the body where tissue (e.g., fat) is found.

[0022] The term "pharmaceutical agent" has a meaning known to those skilled in the art, whether used as an ingredient in a biological scaffold or in any method of using a biological scaffold, and includes at least the following activators: growth factors, differentiation factors, cytokines, chemokines, adipokines, antibiotics, antifungal agents, anti-inflammatory agents, and anticancer agents (chemotherapeutic agents).

[0023] This disclosure provides a biological scaffold comprising at least two components, namely, mammalian platelet lysate and adipose tissue-derived cell fraction (ATDCF). In relevant embodiments, the biological scaffold comprises adipose tissue-derived cells, including, but not limited to, one or more of the following: stromal vascular fraction (SVF) cells, adipose-derived stromal cells, adipose-derived stem cells (collectively referred to herein as "ASCs"), bone marrow-derived mesenchymal stromal cells, and bone marrow-derived mesenchymal stem cells (collectively referred to herein as BM-MSCs).

[0024] As used herein, the term “interstitial vascular fraction (SVF) cells” refers, by definition, to a heterogeneous population of cells isolated from adipose tissue after enzymatic or mechanical disturbance / digestion with exemplary proteases, such as collagenase type 1, or other related enzymes that disturb the extracellular matrix. These SVF cells are not adherent to plastics and do not expand / proliferate in vitro. This population includes, among other things, pre-adipocytes, stromal cells / stem cells, lymphocytes, myeloid cells, fibroblasts, pericytes, endothelial progenitor cells, and hematopoietic stem cells.

[0025] Adipose-derived stromal / stem cells (ASCs): This relatively homogeneous cell population was isolated from adipose-derived SVF cells after adhesion to plastic tissue plates and in vitro culture growth in the presence of growth factors / culture media. This population is relatively abundant in adherent preadipocytes, stromal / stem cells, fibroblasts, pericytes, and endolymphoid progenitor cells, but is depleted in the lymphopeopoietic lineage. These ASCs are pluripotent and can differentiate into adipocytes, chondrocytes, and osteoblasts.

[0026] Bone marrow-derived mesenchymal stromal / stem cells (BM-MSCs): This relatively homogeneous cell population was isolated from bone marrow aspirates after adhesion to plastic tissue plates and in vitro culture and growth in the presence of growth factors / culture media. These BM-MSCs exhibit a phenotypic similarity to ASCs, which possess pluripotent differentiation potential (adipocytes, chondrocytes, osteoblasts).

[0027] A. Biological scaffold In some embodiments, the present disclosure provides a biological scaffold that can be enhanced in various cell types and various growth factors, cell differentiation factors, and other agents useful in the study of cell proliferation, such as inhibitors of tumor or cancer growth. In various embodiments, the biological scaffold comprises at least two components. The first component is a mammalian platelet lysate. The term "mammalian platelet lysate" may include lysates formed from platelets isolated from any mammalian source, including humans.

[0028] The mammalian platelet lysate may include platelet lysates prepared from any natural mammalian platelets or from cultured platelets. In various embodiments of this disclosure, the mammalian platelet lysate may include the use of human platelet lysate for use herein in various products and the use of the scaffolds described herein. In various embodiments, in compositions, products and methods of use, the platelets used in the preparation of the mammalian platelet lysate for use in the disclosed biological scaffolds are human platelets. The mammalian platelet lysate may be prepared in liquid form that can be cryopreserved as a freezeable material to be thawed immediately before use, but the product may also be processed and stored using alternative methods and physical forms (as outlined below), including, but not limited to, lyophilization, micronization, solidification, or combination products. An exemplary form of mammalian platelet lysate is a liquid product derived from human platelet lysate. Sterile, expired human platelets collected by an AABB-authorized blood collection center are received frozen at -20°C for laboratory processing. All processes involving exposure of platelets to air are carried out in a biologically safe cabinet (BSL2). These platelets are subjected to three consecutive freeze / thaw cycles, alternating between room temperature (15°C–25°C) and -80°C. After the final thaw cycle, platelets obtained from 1–50 donors are pooled and centrifuged at 8,000 × g (rcf). Following centrifugation, the apical white layer containing the platelet membrane is aspirated. The remaining clear infiltrate corresponding to this platelet lysate contains undiluted mammalian platelet lysate.

[0029] The second component of the biological scaffold described herein comprises adipose tissue-derived cell fraction (ATDCF). This term refers to at least one cell type derived from adipose tissue. In some embodiments, the biological scaffold of this disclosure comprises at least one cell type from the group consisting of stromal vascular fraction (SVF) cells, adipocyte-derived stromal cells, adipose-derived stem cells (collectively referred to herein as "ASCs"), bone marrow-derived mesenchymal stromal cells, and bone marrow-derived mesenchymal stem cells (collectively referred to herein as BM-MSCs). In each example, the ATDCF comprises at least one of the aforementioned cell types, and the ATDCF is derived from adipose tissue. The biological scaffolds of this disclosure are adapted to specific uses by supplementing the mammalian platelet lysates with one or more of these cell types, optionally one or more agents (e.g., growth factors, differentiation factors, cytokines, chemokines, adipokines, antibiotics, antifungal agents, anti-inflammatory agents, anticancer agents (such as chemotherapeutic agents)) to provide biological scaffold-containing formulations, differentiation constructs, grafts, wound dressings, and other uses disclosed herein.

[0030] B. Products incorporating a biological scaffold Kits for cell differentiation In some embodiments, the biological scaffolds described herein can be used as physician-grade starting materials to prepare specific differentiated cells and tissues. In some exemplary embodiments, these kits can be used for research purposes. For example, an exemplary kit may include a container containing mammalian platelet lysate, a multiwell substrate, and Reagent A (consisting of Dulbecco's Modified Essential Medium, supplemented with 1% antibiotic / antifungal agent and other relevant cell culture media containing nutrients, amino acids, and growth factors necessary for cell culture and white or brown adipocyte differentiation). In certain embodiments, if the user wishes to prepare differentiated cells in a three-dimensional (3D) cell culture, the 3D tissue culture to be used may be prepared according to the following exemplary steps. Prepare a mammalian platelet lysate supplement medium by adding 25% (v / v) of the mammalian platelet lysate to 1% antibiotic / antifungal agent and 74% Dulbecco's Modified Essential Medium, or other relevant cell culture medium containing nutrients, amino acids, and growth factors necessary for cell culture. Do not warm the mixture in a water bath after mixing. Maintain at room temperature. Thaw these cells (SVF cells, and / or ASCs, and / or BM-MSCs) in a 37°C water bath from liquid nitrogen until the ice disappears. Wash each cryovial once with 1 mL of standard Stromal Medium. Centrifuge the tubes at 300 × g (1200 rpm) for 5 minutes. Divide the cell pellet into 5 × 10⁶ 5 The cells were resuspended in hPL supplement medium at a concentration of cells / mL. Immediately, the cell / hPL mixture was plated. When placed in a CO2 incubator, the mixture immediately gelled. The evaporated medium was replaced every 7 days to maintain 3D culture. The old medium of the culture was removed by aspirating with a 1 mL micropipette. Half the volume of hPL-medium was added to the upper corner of the gel to replace it with fresh medium.

[0031] In another relevant embodiment, differentiated adipocytes may be induced in the biological scaffold of this disclosure. In an exemplary method, the user may utilize mammalian platelet lysate, a substrate for culturing white or brown adipocytes / tissues, and reagent A. Prepare the 3D tissue culture according to the following instructions: Prepare mammalian platelet lysate supplement medium by adding 25% (v / v) of the mammalian platelet lysate to 1% antibiotic / antifungal agent and 74% Dulbecco's Modified Essential Medium, or other relevant cell culture medium containing the nutrients, amino acids, and growth factors necessary for cell culture. After mixing, do not warm in a water bath. Maintain at room temperature. Thaw the stromal vascular fraction (SVF), bone marrow-derived mesenchymal stem / stroma (BM-MSC), or adipocyte-stroma / stem (ASC) cells in a cryovial from liquid nitrogen in a 37°C water bath until the ice disappears. Wash each cryovial once with 1 mL of standard Stromal Medium. Centrifuge the tubes at 300 × g (1200 rpm) for 5 minutes. Divide the resulting cell pellet into 5 × 10 5 The cells were resuspended in hPL supplement medium at a concentration of cells / mL. Immediately, the cell / hPL mixture was plated. When placed in a CO2 incubator, the mixture immediately gelled. The evaporated medium was replaced every 7 days to maintain 3D culture. The old medium of the culture was removed by aspirating with a 1 mL micropipette. Half the volume of hPL-medium was added to the upper corner of the gel to replace it with fresh medium.

[0032] In related embodiments, a kit is provided for differentiating progenitor cells into adipose tissue or brown / beige adipose tissue. An exemplary kit may include one or more cryovials of adipose tissue-derived SVF cells, one or more 100 ml bottles of culture media from LaCell LLC (LaCell LLC, New Orleans, Louisiana, USA) products specific to cell proliferation (StromaQual), lipidogenesis (AdipoQual), chondroogenesis (ChondroQual), or osteoblast differentiation (OsteoQual), and mammalian platelet lysates in liquid form, which can be combined with tissue culture plates (6, 12, or 24 wells) suitable for sterile cell proliferation and differentiation. The kit may be further modified by incorporating proprietary lipidogenesis (or) differentiation media designed to selectively promote white adipose tissue (or brown / beige adipose tissue).

[0033] In related embodiments, kits for cell proliferation (StromaQual), lipidogenesis differentiation (AdipoQual), chondroogenesis differentiation (ChondroQual), or osteoblast differentiation (OsteoQual) are provided. An exemplary kit may include a frozen tube of mammalian platelet lysate in liquid form, one or more cryovials of adipose tissue-derived ASC cells, one or more 100 ml bottles of culture medium from LaCell LLC products specific to cell proliferation (StromaQual), lipidogenesis differentiation (AdipoQual), chondroogenesis differentiation (ChondroQual), or osteoblast differentiation (OsteoQual), and tissue culture plates (6, 12, or 24 wells) suitable for sterile cell proliferation and differentiation. The kit may be further modified by incorporating proprietary lipidogenesis differentiation media designed to selectively promote white adipose tissue or brown / beige adipose tissue.

[0034] In related embodiments, kits are provided for cell proliferation (StromaQual), lipidogenesis differentiation (AdipoQual), chondroogenesis differentiation (ChondroQual), or osteoblast differentiation (OsteoQual). An exemplary kit may comprise a frozen tube of mammalian platelet lysates in liquid form, one or more cryovials of BM-MSCs derived from bone marrow aspirates, one or more bottles of culture medium from LaCell LLC products specific to cell proliferation (StromaQual), lipidogenesis differentiation (AdipoQual), chondroogenesis differentiation (ChondroQual), or osteoblast differentiation (OsteoQual), and tissue culture plates (6, 12, or 24 wells) suitable for sterile cell proliferation and differentiation. The kits described herein may be further modified by incorporating proprietary lipidogenesis differentiation media designed to selectively promote white adipose tissue differentiation or brown / beige adipose tissue differentiation.

[0035] In exemplary embodiments, exemplary biological scaffolds are provided that can be used without modification for tissue reconstruction or cosmetic purposes, or that can be applied to solid or semi-solid surfaces for introduction into or adjacent to tissue defects, such as wounds or damaged bone. In some embodiments, the biological scaffold may comprise a solution of mammalian platelet lysate, such as human platelet lysate, which is directly mixed with adipose tissue-derived SVF cells as a combination product that can be used as an injectable product, prepared using the method herein, or processed as a solid or semi-solid scaffold that can be implanted into tissue defects, such as bone, for tissue repair. Similarly, the biological scaffold may be inserted into or applied to at least one surface of orthopedic devices, such as bone cages, bone screws, pedicles, and other orthopedic devices that are in contact with the patient's bone to at least some extent. In other embodiments, sections or portions of affected bone may be treated using a solid substrate containing or coated with the biological scaffold, or an exemplary biological scaffold described herein. In non-limiting examples, osteonecrosis of the jaw or femoral head may be treated by excising the affected bone tissue, then transplanting autologous or allogeneic bone tissue, and at least one surface of the transplanted bone may be coated with or contain an exemplary biological scaffold, such as a biological scaffold containing human platelet lysate and BM-DSC. In exemplary osteonecrosis repair of the femoral head, several initial treatments can be designed using the biological scaffolds of this disclosure. For example, joint-preserving surgery may include core decompression of the femoral head, which may optionally be combined with autologous bone marrow cell transplantation, autologous bone grafting with vascularized muscle pedicle or vascular anastomosis, or non-vascularized autologous bone grafting, and osteotomy.

[0036] In some embodiments, core decompression can reduce pain. In some embodiments, a 3 mm diameter drill can be used to create multiple holes in the femoral head, and then the biological scaffold of the present disclosure can be filled.

[0037] In some embodiments, autologous bone marrow cell transplantation may be used. In these embodiments, the surgeon extracts more than 200 mL of iliac bone marrow blood, isolates mononuclear cells in vitro (without culture medium), and then simply injects or transplants them with the biological scaffold of the present disclosure. Other exemplary embodiments may involve the use of autologous bone grafting procedures relating to the use of the biological scaffold of the present disclosure to coat an angiogenic muscular peduncle, vascular anastomosis, or non-angiogenic autologous bone graft, wherein at least a portion of the surface of the graft is coated with the biological scaffold. Examples of angiogenic bone grafts include deep iliac bone, superficial iliac vein grafts, lateral circumflex femoral branch of the greater trochanter, and gluteal branch of the greater trochanter. Bone grafts with muscular peduncles typically utilize the quadratus femoral muscle.

[0038] Allogeneic or autologous fibrous grafts may be used in surgical procedures to coat or fabricate bone grafts to include the biological scaffolds of this disclosure.

[0039] Impact bone grafting may utilize autologous or allogeneic bone coated with the biological scaffold of this disclosure, with or without the use of bone morphogenetic proteins known in the art, as well as other bone conduction and bone induction agents. These bone grafting procedures include exemplary biological scaffolds containing human platelet lysate and BM-DSC. Osteotomy most commonly takes the form of rotational femoral neck osteotomy, such as greater trochanter or inverter osteotomy.

[0040] In other embodiments, most patients with osteonecrosis of the femoral head will ultimately undergo arthroplasty. With advances in the design, materials, and techniques of artificial joints, the scope of joint-preserving surgery has narrowed, while the indications for arthroplasty have expanded. The type of artificial joint can be selected according to the following recommendations: 1. Due to the complexity associated with the metal-on-metal bearing surface, surface reshaping plays only a limited role in ONFH cases. In each of these arthroplasty procedures, the artificial joint is coated with a biological scaffold of the present disclosure, for example, an exemplary biological scaffold containing human platelet lysate and BM-DSC.

[0041] In relevant embodiments, the exemplary biological scaffolds of this disclosure provide cell constructs containing adipose-derived stromal cells and / or adipose-derived stem cells. In various relevant embodiments, the ASC-containing biological scaffolds include, in the form of a liquid formulation, a combination product in which a mammalian platelet lysate, e.g., human platelet lysate prepared according to the method herein, can be used as an injectable agent or processed as a solid or semi-solid scaffold for tissue repair, directly mixed with adipose tissue-derived ASCs.

[0042] In related embodiments, the exemplary biological scaffolds of this disclosure can be used as injectable agents or as combined products that can be processed as solid or semi-solid scaffolds for tissue repair, providing cell constructs containing bone marrow aspirate-derived BM-MSCs.

[0043] In various exemplary embodiments, this disclosure provides a kit or package that can be stored and transported at -20°C or -80°C, comprising a freezing tube of a mammalian platelet lysate liquid formulation, one or more cryovials of adipose tissue-derived SVF cells, one or more bottles of a medium from LaCell LLC (LaCell LLC, New Orleans, Louisiana) specific for cell proliferation (StromaQual), lipidogenesis differentiation (AdipoQual), chondroogenesis differentiation (ChondroQual), or osteoblast differentiation (OsteoQual), and a tissue culture plate (6, 12, or 24 wells) suitable for sterile cell proliferation and differentiation. The exemplary kits described herein may be further modified by incorporating proprietary lipidogenesis differentiation media designed to selectively promote white adipose tissue or brown / beige adipose tissue.

[0044] In various exemplary embodiments, the Disclosure provides a kit or package that can be stored and transported at -20°C or -80°C, comprising a freezing tube for a mammalian platelet lysate liquid formulation, one or more cryovials of adipose tissue-derived ASCs, one or more bottles of a culture medium from LaCell LLC products specific to cell proliferation (StromaQual), lipidogenesis differentiation (AdipoQual), chondroogenesis differentiation (ChondroQual), or osteoblast differentiation (OsteoQual), and a tissue culture plate (6, 12, or 24 wells) suitable for sterile cell proliferation and differentiation. The exemplary kit may be further modified by incorporating proprietary lipidogenesis differentiation media designed to selectively promote white adipose tissue or brown / beige adipose tissue.

[0045] In various exemplary embodiments, the Disclosure provides a kit or package that can be stored and transported at -20°C or -80°C, comprising a frozen tube of a mammalian platelet lysate liquid formulation, one or more cryovials of BM-MSCs derived from bone marrow aspirates, one or more bottles of culture medium from LaCell LLC products specific to cell proliferation (StromaQual), lipidogenesis differentiation (AdipoQual), chondroogenesis differentiation (ChondroQual), or osteoblast differentiation (OsteoQual), and a tissue culture plate (6, 12, or 24 wells) suitable for sterile cell proliferation and differentiation. The kits of the Disclosure may be further modified by incorporating proprietary lipidogenesis differentiation media designed to selectively promote white adipose tissue or brown / beige adipose tissue differentiation.

[0046] In various exemplary embodiments, the Disclosure provides a kit or package that can be stored and transported at -20°C or -80°C, comprising only a freezing tube of a mammalian platelet lysate liquid formulation and a package of desalted bone powder, or hydroxyapatite / tricalcium phosphate, or equivalent osteoinductive / osteoconductive bone powder, or further comprising one or more cryovials of SVF cells and / or one or more cryovials of ASCs and / or one or more cryovials of BM-MSCs. The contents of the kit are combined by an orthopedic surgeon, cosmetic surgeon, or craniofacial surgeon in a clinical setting or operating room and delivered to the patient's surgical or injury site as a mixture with a paste or solidified material that promotes bone regeneration.

[0047] In some embodiments, a kit is provided that can be stored and transported at -20°C or -80°C, comprising a cryovial tube for mammalian platelet lysates in liquid formulation form, and a nozzle device comprising, or not comprising, cryovials of SVF cells and / or ASCs and / or BM-MSCs. The resulting combined product, comprising a liquid biological scaffold, is designed for spray delivery to the skin or wound site of patients with chemical, electrical, radioactive, or thermal burns or injuries. The product of the kit can be delivered as mammalian platelet lysates alone, or in combination with SVF cells and / or ASCs and / or BM-MSCs.

[0048] In some embodiments, a product comprising the biological scaffold of the present disclosure may include a package comprising a sterile bandage, synthetic mesh, or gauze material, and the biological scaffold of the present disclosure. In exemplary cases, the bandage, mesh, or gauze is pre-soaked in mammalian platelet lysate and coated during the manufacturing process, and subsequently stored as a freeze-combined product or as a freeze-dried product that can be stored for extended periods at room temperature or 4°C. When in use, the bandage, mesh, or gauze can then be coated with ATDCF cells applied to tissue requiring coverage of the bandage, mesh, or gauze material. For example, for cartilage or muscle tissue, the mesh can be impregnated with mammalian platelet lysate, and the tissue to be fixed to the mesh during surgery can be added to a specific cell fraction that is operable to guide the mesh into the tissue, and the mesh can be contacted, for example, in hernia repair and other abdominal and genitourinary applications, where the mesh can be fixed to muscle or cartilage material. Alternatively, the bandage or gauze material and mammalian platelet lysate may be transported separately in packages, allowing a physician or surgeon to combine them directly with ATDCF in the clinical setting. The bandage is then administered directly to the wound site or the patient's wound.

[0049] In some embodiments, the disclosure provides a package containing a cryopreservation container for mammalian platelet lysates and one or more cryovials of ATDCF cells, e.g., SVF cells, and / or ASCs, and / or BM-MSCs. These materials are transported to the user using dry ice and stored at -20°C or -80°C before use. These materials are designed to be used with a 3D printer to enable the fabrication of customized three-dimensional cell / scaffold structures. In some embodiments, the same biological scaffold material, or different biological scaffold materials, may be engraved onto different regions, sections, and / or surfaces.

[0050] C. Exemplary use of biological scaffolds In some embodiments, mammalian platelet lysates can be combined with human adipose tissue-derived stromal vascular fraction (SVF) cells, adipose-derived stromal / stem cells (ASCs), and / or bone marrow-derived mesenchymal stromal / stem cells (BM-MSCs) to create combination products suitable for studying adipose tissue or bone and bone marrow biology. These combination products can be cultured in vitro in or out of the presence of cocktails known to promote lipidogenesis (AdipoQual), chondrogenesis (ChondroQual), or osteoogenesis (OsteoQual). When mammalian platelet lysates are combined with SVF cells, ASCs, or BM-MSCs in the presence of AdipoQual, they form three-dimensional adipose tissue structures and functions that can be evaluated based on gene, protein, and small molecule expression, as well as glucose uptake, lipolysis, metabolic activity, and other adipose tissue function assays. In the presence of ChondroQual, when mammalian platelet lysates are combined with SVF cells, ASCs, or BM-MSCs, they form a three-dimensional cartilage tissue structure and function that can be evaluated based on gene, protein, and small molecule expression, as well as glycosaminoglycan production, metabolic activity, and other cartilage tissue function assays. In the presence of OsteoQual, when mammalian platelet lysates are combined with SVF cells, ASCs, or BM-MSCs, they form a three-dimensional bone and bone marrow tissue structure and function that can be evaluated based on gene, protein, and small molecule expression, as well as other bone tissue function assays such as extracellular matrix desalting, calcium uptake, metabolic activity, and lymphopoeia.

[0051] In some embodiments, the biological scaffolds of the present disclosure can be used as combination kit products for the generation, long-term culture, and proliferation of human adipose tissue-derived cells for research purposes in normal or diseased adipose tissue. The biological scaffolds can be initiated and cultured at 37°C to produce adipocyte-derived three-dimensional scaffolds. In various embodiments, the present disclosure provides kits that may include component I necessary for independent setup, maintenance, and assay endpoints for evaluating adipose tissue gene, protein, and small molecule expression, as well as tissue functionality. In some embodiments, the kits of the present disclosure may include a transport package containing cryopreserved primary ATDCF cell vials, mammalian platelet lysates, culture media, substrates, and instructions. In some embodiments, white or brown / beige adipose-differentiated tissue bioscaffolds can be customized to include SVF cells, and / or ASCs, and / or BM-MSCs, as well as media specifically designed to promote the formation and differentiation of white or brown / beige adipose tissue depots.

[0052] In the relevant embodiments, white and / or brown / beige adipose-differentiated tissue bioscaffolds can be customized as a combination kit product using SVF cells and / or ASCs and / or BM-MSCs, or offered as a contract service for drug discovery research purposes in normal or diseased adipose tissue. The cells used can be obtained from healthy donors or donors with a specific disease or condition of interest, such as obesity or type 2 diabetes (T2DM). The white and / or brown / beige adipose-differentiated tissue bioscaffold combination product / service can be cultured at 37°C in or without pharmaceuticals or medicinal cosmetics, and the early and late responses of the tissue can be measured. The assay endpoint can be measured to evaluate adipose tissue gene, protein, and small molecule expression, as well as cellular biological, chemical, toxicological, and functional responses to drugs. In some exemplary embodiments, a white and / or brown / beige adipose-differentiated tissue bioscaffold may include a transport package containing cryopreserved primary cell vials, mammalian platelet lysates, culture medium, substrate, and instructions. The service may consist of setting up and maintaining adipose tissue culture and adding compounds to treat disease conditions. The service may include evaluating the response of adipose tissue to chemical compounds by assay.

[0053] A biological scaffold product for creating humanized mouse fat depot models.

[0054] In some embodiments, the biological scaffold can be used to create a mouse model of human adipose tissue or adipose depot. The biological scaffold (containing human SVF cells and / or human ASCs) can be subcutaneously injected into the epidermis / dermis of either immunonormal or immunodeficient mice in a volume of 250 μl or more using a sterile syringe and a 19-gauge needle. The subcutaneous injection site can be sealed with Vetbond or an equivalent material to prevent subsequent leakage. The grafts are cured for up to 12 weeks. During this period, the grafts acquire the characteristics of a differentiated adipose depot. The human-derived cells constitute the proportion of cells in these adipose depots over a period of up to 12 weeks or more. The mice can be fed a diet with a fat content of 45% or more (high-fat diet) to enhance the enlargement and differentiation of the humanized adipose depot. Similarly, mice can be treated with small molecule chemicals or peptides / proteins to evaluate their effects on the metabolism of human adipose tissue under physiological conditions. These humanized fat depots can be monitored using standard scientific methods, including, but not limited to, cell biological methods, histological methods, in vivo imaging methods, metabolic methods, and related methods.

[0055] The biological scaffolds of this disclosure may be used in vivo or in vitro to create patient-derived xenografts or tumors.

[0056] Human tumors can be cultured and proliferated using a biological scaffold containing human adipose tissue-derived SVF cells or ASCs. The biological scaffold of this disclosure can be used with tumor cell lines or tumor fragments (1-10 mm). 3By combining (volume) and culturing in vitro at 37°C, a three-dimensional tumor-containing scaffold containing adipocytes can be produced. This combined biological scaffold formulation then provides a stromal / vascular environment that promotes long-term survival, growth, and proliferation of human-derived primary or metastatic tumors acquired before or after patient exposure to chemotherapy, radiotherapy, or immunotherapy. 1-10 mm 3 Tumor fragments are either transplanted directly into a combined adipocyte biological scaffold in vitro, or injected subcutaneously into immunodeficient mice in liquid form using a biological scaffold. These tumors are maintained either in vitro or in vivo for a period of 1 to 12 months, and their growth is monitored based on volume, size, cell count, and factor secretion. These tumors originate from sources of breast cancer, prostate cancer, bone cancer, colorectal cancer, or other malignancies.

[0057] In some embodiments, the biological scaffolds of this disclosure may be used in mouse models in vitro or in vivo for the development of human bone or human bone marrow.

[0058] Using biological scaffolds containing BM-MSCs, either using desalted bone powder, hydroxyapatite / tricalcium phosphate, or equivalent osteoinducible / osteoconductive bone powder, or none of these, in vitro or in vivo constructs suitable for human hematopoiesis and bone formation studies can be prepared. These biological scaffolds can be cultured in vitro in the presence of cytokines and growth factors known to promote lymphoporesorption, and the detection and proliferation of non-adherent and adherent cells expressing surface antigens related to the lymphoid and myeloid systems can be monitored. Alternatively, the combination product can be transplanted into an empty bone marrow cavity or subcutaneously into immunodeficient mice, and the production of human hematopoietic and osteogenic cells can be monitored by flow cytometry and immunohistochemical or molecular biological analysis of hard tissue. The resulting in vitro and in vivo models can be used to design experiments on human blood and bone formation.

[0059] Biological scaffolds containing one or more ATDCF cells, e.g., SVF cells, and / or ASCs, and / or BM-MSCs, can be used for bone development in clinical settings. In some embodiments, orthopedic surgeons, plastic surgeons, or craniofacial surgeons can use biological scaffolds containing one or more ATDCF cells, e.g., SVF cells, and / or ASCs, and / or BM-MSCs, to repair traumatic, congenital, or surgical bone defects in weight-bearing and non-weight-bearing areas. A combination of a biological scaffold containing ATDCF (SVF cells, and / or ASCs, and / or BM-MSCs) and an osteoinductive / osteoconductive ceramic material can be combined in clinical settings to create a paste that is delivered to the bone defect site. The surgeon can then implant and contain this combined material within the dimensions of the defect using surrounding tissue or appropriate conduits or membranes. The surgeon stabilizes the defect using an open reduction / internal fixation method appropriate for standard treatment. Subsequently, the aforementioned combined biological scaffold promotes and accelerates bone repair, recovery, and remodeling during the postoperative period, which is monitored non-invasively using radiographic imaging.

[0060] In some embodiments, the biological scaffolds of the present disclosure may be formulations to be sprayed for the treatment of burns occurring after chemical, electrical, flame, radiation, or thermal injuries.

[0061] In some embodiments, a biological scaffold formulated for administration by spray can be used by general surgeons, plastic surgeons, and reconstructive surgeons, as well as trauma surgeons, to treat skin with first-degree, second-degree, or third-degree burns and underlying tissues in acute and chronic conditions. The spray formulation containing the biological scaffold may include an operable nozzle that delivers only the biological scaffold as a controlled spray of small droplets / particles to the injured area from a distance of 10 cm or less from the surface. The surgeon or other medical professional providing the service can monitor the degree of wound surface coverage based on visual inspection. Where appropriate, the spray formulation of the biological scaffold and the device for applying the biological scaffold can deliver autologous or allogeneic cells, including, but not limited to, SVF cells, and adipose tissue-derived ASCs, bone marrow-derived BM-MSCs, or, more optionally, skin-derived dermal fibroblasts, epidermal cells, and / or keratinocytes. With or without cell supplementation, the application of the sprayed biological scaffold is designed to promote and enhance the formation of intact new skin, including pigment epithelial cells, hair follicles, sebaceous glands, sweat glands, immune cells, and the underlying tissue structures necessary to maintain all functions that result in a proper epidermis / dermis and a healthy outer layer, such as maintaining fluid evaporation and preservation, immune barriers, vitamin D metabolism, and UV protection.

[0062] Various formulations, products, and applicators containing the biological scaffold of this disclosure may be used for the treatment of pressure ulcers, trauma, or diabetic ulcers.

[0063] In various embodiments, biological scaffolds, formulated as gels or solutions and applied to semi-solid or solid substrates such as bandages, gauze, and mesh, can be used individually or in combination by general surgeons, plastic surgeons, and reconstructive surgeons to treat acute and chronic skin wounds involving the epidermis, dermis, and / or underlying tissues (fat, skeletal muscle, bone), such as compression trauma or pressure ulcers resulting from ischemic / reperfusion injury in elderly and paraplegic / quadriplegic patients, or diabetic ulcers resulting from ischemia due to impaired neovascularization of the distal extremities in patients with either type 1 or type 2 diabetes. Before applying the biological scaffold, the wound may be debridemented, and the surrounding skin surface may be cleansed with 70% ethanol and betadine or an equivalent antimicrobial cleanser. The mammalian platelet lysate or biological scaffold-containing preparation may be injected directly into the base of chronic skin wounds / pressure ulcers / diabetic ulcers using a #19 or #21 gauge needle in a fan-shaped manner covering the entire surface of the wound, under sterile techniques. The injection should be made so that it enters the skin from the outside surrounding the wound site itself to avoid the risk of contamination. The mammalian platelet lysate or biological scaffold-containing injection may be repeated every other week, monthly, or at infrequent intervals, based on the surgeon's clinical judgment. Furthermore, the mammalian platelet lysate or biological scaffold-containing preparation, for example, the mammalian platelet lysate or biological scaffold, or a product containing the biological scaffold, such as a bandage, mesh, or dressing, may be delivered or placed on the surface of the debridement pressure ulcer or diabetic ulcer injury site. Next, the area is covered with a sterile Adaptic or Tegaderm bandage to isolate the gelled product or the bandage from the surrounding area. Based on the surgeon's judgment, this topical therapy can be repeated up to three times a week. Furthermore, this topical therapy can be delivered in combination with negative pressure wound vacuum, according to current standard care practices.The application of mammalian platelet lysates, biological scaffolds, or products containing such biological scaffolds, such as bandages, meshes, or dressings, enhances and amplifies functional efficacy indicators such as the rate of closure and healing of chronic skin wounds / ulcers based on direct observation and histological structure, as well as skin health, moisture loss / evaporation, immune barrier, and appearance.

[0064] In the first embodiment, a biological scaffold is formed by mixing mammalian (e.g., human) platelet lysate with one or more ATDCF cells. In some embodiments, a biological scaffold is formed by mixing one or more ATDCF cells, e.g., stromal vascular fraction (SVF) cells, and / or adipose-derived stromal / stem cells (ASCs), and / or bone marrow-derived mesenchymal stromal / stem cells (BM-MSCs), selected from SVF cells, and / or ASCs, and / or BM-MSC cell types. An exemplary procedure for preparing such a biological scaffold may use the following steps: Unless otherwise noted, all steps should be performed in a biologically safe cabinet. The desired amount of concentrated, expired human platelet solution is placed in a 15 mL or 50 mL conical tube. If not immediately for use in 3D cell culture, the solution is stored at -20°C after three freeze / thaw cycles before preparing the human platelet lysate preparation. Perform three freeze / thaw cycles and lyse the platelets by osmosis. This releases growth factors for cell culture in maximum yield. Centrifuge at 8,000xg for 20 minutes at room temperature. Remove the solid (white) top layer of human platelet membrane by aspirate. The remaining liquid infiltrate contains human platelet lysate. Prepare a human platelet lysate supplement medium by adding 7.5% (v / v) human platelet lysate to 1% antibiotic / antifungal agent and 91.5% Dulbecco's Modified Essential Medium, or other relevant cell culture medium containing essential nutrients, amino acids, and growth factors for cell culture. Note: The percentage concentration of human platelet lysate can be changed by appropriately adjusting the corresponding content of DMEM or other relevant cell culture medium while continuing to supplement with 1% antibiotic / antifungal agent. Do not warm in a water bath after mixing. Maintain at room temperature. The prepared human platelet lysate is combined with a cell population or a mixture of cell populations to activate gelation (formation of a thick matrix) that promotes cell secretion, proliferation, and differentiation of the extracellular matrix. After mixing the cells with the human platelet lysate, the plate is placed in an incubator for 20 minutes to activate gelation.The process continues with proliferation, differentiation, transplantation into mice, or other endpoints for measurement.

[0065] Biological scaffold for use in tissue regeneration.

[0066] In some embodiments, the biological scaffolds of this disclosure may be used for replacement, tissue reconstruction, expansion and augmentation, and cosmetic applications. Exemplary methods may include a surgeon or physician selecting a product incorporating the biological scaffolds described herein to treat patients with trauma, surgical or tumor resection, and those with scars or defects resulting from congenital anomalies such as Poland syndrome or Tretcher-Collins syndrome. In one such example, the surgeon may use a cryopreservation container (5 or 10 mL) stored at -20°C containing mammalian platelet lysates manufactured under current Good Manufacturing Practice (cGMP) and approved for clinical use. In some embodiments, the platelet lysates are human platelet lysates. However, platelets from other mammalian species may be used, and the human platelets may be autologous or allogeneic. In some embodiments, mammalian (e.g., human) platelet lysates can be used alone or in combination with autologous patient-derived stromal vascular fraction (SVF) cells isolated from liposuction tissue harvested at the time of treatment, using a closed apparatus or device such as those manufactured by Tissue Genesis (Honolulu HA) or Cytori Therapeutics (San Diego CA). Alternatively, the human platelet lysates may be replaced in the form of a liquid formulation directly mixed with adipose tissue-derived ASCs as a combination product, which can be used as an injectable material or processed as a solid or semi-solid scaffold for tissue repair.

[0067] In several relevant embodiments, the biological scaffold for use in tissue reconstruction or expansion may be used as an injectable material or as a combination product that can be processed as a solid or semi-solid scaffold for tissue repair, containing human platelet lysates in the form of a liquid formulation directly mixed with BM-MSCs derived from bone marrow aspirates, and allogeneic complex cell therapies can be created using allogeneic adhesive adipose tissue or bone marrow-derived cells cultured and grown under current Good Manufacturing Practice (cGMP) protocols. A combination of liquid human platelet lysates supplemented with or without autologous or allogeneic cells may be used by a surgeon or physician as an injectable material. The physician draws the liquid human platelet lysates into a sterile syringe with or without autologous or allogeneic cells (SVF cells, and / or ASCs, and / or BM-MSCs). This liquid material is then subcutaneously injected into the subcutaneous layer beneath the tissue to be treated, e.g., a wound, scar, or skin or tissue defect. This liquid is delivered in a fan-shaped pattern from the injection site using a #19 or #21 gauge needle, allowing it to penetrate thoroughly into the dermis below. The physician injects a sufficient amount to achieve the desired cosmetic effect with respect to the contour of the wound or defect. This procedure can be performed once or repeated multiple times to achieve the desired cosmetic and reconstructive results.

[0068] In an exemplary embodiment, the mammalian platelet lysate and / or biological scaffold may be used for tissue remodeling and cosmetic improvement. Exemplary methods may include: providing the mammalian platelet lysate to a surgeon, operating room, or other health delivery center as cryopreserved aliquots containing 1 to 20 ml of mammalian platelet lysate in accordance with FDA and cGMP guidelines. Transparent containers are suitable as they do not pose a risk of breakage and can withstand freeze / thaw cycles. Thawing the mammalian platelet lysate container rapidly in a 37°C bath or dry oven until most of the ice crystals have disappeared. Opening the mammalian platelet lysate container in the operating room and drawing the liquid contents into a sterile 1 to 20 ml syringe. If applicable, the surgeon may similarly thaw and draw up cryopreserved vials of allogeneic SVF cells, ASCs, or BM-MSCs. Alternatively, the surgeon may collect liposuction from the patient themselves as a source of autologous SVF cells. Next, either autologous or allogeneic cells are directly mixed with mammalian platelet lysate by connecting two syringes via a sterile stopcock to create a combined biological scaffold product. Then, using a needle of #19 gauge or smaller, the surgeon injects the mammalian platelet lysate or biological scaffold product into the patient using a modified Coleman technique, injecting small amounts of liquid as the needle is withdrawn and delivering it in a fan-like manner to the entire subcutaneous tissue. The surgeon can deliver the mammalian platelet lysate or biological scaffold product by injection into subcutaneous and supermuscular breast tissue to achieve volume increase and breast enlargement for cosmetic purposes, deliver it to the base of scars or unsightly areas on the face or elsewhere to eliminate volume deficiency and improve appearance, or deliver it to the base of excised pressure ulcers or diabetic ulcers to promote wound suturing and healing.

[0069] Application of biological scaffolds to drug discovery

[0070] In some embodiments, the biological scaffolds described herein may be used to prepare cell constructs useful for drug discovery. In one exemplary embodiment, the cell construct containing the biological scaffold may include mammalian platelet lysate in liquid or semi-solid (gelatinous) or solid form (scaffold applied to a solid substrate), a substrate for culturing white or brown fat, and a container of reagent A. An exemplary method for using such a cell construct for drug discovery may include the following steps: Prepare a 3D tissue culture using the following instructions: Prepare a mammalian platelet lysate supplement medium by adding 25% (v / v) of the mammalian platelet lysate to 1% antibiotic / antifungal agent and 74% Dulbecco's Modified Essential Medium, or other relevant cell culture medium containing nutrients, amino acids, and growth factors necessary for cell culture. After mixing, do not warm in a water bath. Maintain at room temperature. Thaw the stromal vascular fraction (SVF), bone marrow-derived mesenchymal stem / stromal (BM-MSC), or adipose-derived stromal / stem (ASC) cells in cryovials in a 37°C water bath from liquid nitrogen until the ice disappears. Wash each cryovial once with 1 mL of standard Stromal Medium. Centrifuge the tubes at 300 × g (1200 rpm) for 5 minutes. Divide the resulting cell pellet into 5 × 10⁶ cells. 5 The cells were resuspended in hPL supplement medium at a concentration of cells / mL. Immediately, the cell / hPL mixture was plated. When placed in a CO2 incubator, the mixture immediately gelled. The evaporated medium was replaced every 7 days to maintain 3D culture. The old medium from the culture was removed by aspirating with a 1 mL micropipette. Half a volume of hPL-medium was added to the upper corner of the gel to replace it with fresh medium. The desired drug was added directly to the culture at the concentration desired for performing the drug discovery assay. The endpoint of the test was imaging of changes in cell population, protein expression and secretion, gene expression, glucose uptake, lipolysis, or metabolic activity. [Examples]

[0071] D. Examples

[0072] Example 1: Preparation of components for a biological scaffold Isolation of bone marrow mesenchymal stem cells (BM-MSCs) To isolate BM-MSCs, first obtain a bone marrow aspirate that has been anticoagulated with heparin or citrate to prevent coagulation. Before use, maintain the bone marrow aspirate on moist ice at 4°C and perform all steps in an open container in a BSL2 biological safety cabinet. Thoroughly mix Ficoll Paque solution (GE Healthcare® Ficoll-Paque® PREMIUM, 1.078 g / mL). Dispense 20 ml of Ficoll Paque solution into a sterile 50 ml conical tube. Carefully overlay 10 ml of bone marrow aspirate on top of the Ficoll Paque layer and leave without mixing. After sealing the tube, centrifuge at 500 × g for 30 minutes at room temperature without braking using a benchtop Sorvall® Legend® centrifuge. After returning the conical tube to the BSL2 biological safety cabinet, aspirate and remove the clear plasma layer on top and dispense into a new sterile 50 ml conical tube. Next, the buffy coat layer of mononuclear cells is aspirated from the top of the Ficoll Paque layer. The mononuclear cells are transferred to a sterile 15 ml conical tube and suspended in 3 times the volume of sterile Phosphate Buffered Saline (PBS) solution. This tube is centrifuged at 200 × g for 10 minutes at room temperature. After washing away contaminants and the Ficoll-Paque, the supernatant is aspirated from the pelleted mononuclear cells. The pelleted mononuclear cells are resuspended in 1–10 ml of LaCell Stromal Medium™. 10 μl is removed and mixed with an equal volume of LaCell Live Dead Assay Solution™ containing acridine orange / ethidium bromide, and the viable (green) and dead (red) nucleated cells are counted using a fluorescence microscope (Motic AE30 Epifluorescent Microscope). Based on the percentage of viable cells and the total number of cells / milliliter, the total number of nucleated cells is calculated using the following formula.

[0073] Total number of living cells = Percentage of living cells × Volume (ml) × Total number of cells / ml

[0074] 10 5 ~10 6 Suspend mononuclear cells in a concentration of LaCell Stromal Medium® at a concentration of 1 ml / 1 ml. Place 5 × 10 units into T25, T75, or T175 flasks, each with a volume of 15, 25, or 35–50 ml of LaCell Stromal Medium®. 3 ~5 x 10 4 / 1cm 2 Seed the cell suspension at the specified density. Incubate these flasks in a humidified 5% CO2 incubator at 37°C for 1–3 weeks. Every 2–3 days, replace 50% of the medium with fresh LaCell Stromal Medium® or a 1:1 ratio of fresh:BM-MSC acclimatized LaCell Stromal Medium®. Once the BM-MSCs reach 70–90% confluence, subculture the cells by digesting with 0.25% trypsin at 37°C in a humidified 5% CO2 incubator. Monitor adherent cells in the flask using phase-contrast microscopy to confirm that trypsin digestion has released single-cell suspensions. After 5–20 minutes of trypsin digestion, add an equal volume of LaCell Stromal Medium® to stop the enzymatic digestion reaction, and then transfer the total cell volume to a sterile 15 ml or 50 ml conical tube. Remove 10 μl of the cell suspension and mix with an equal volume of trypan blue solution. Determine the total number of cells and the percentage of viable cells by counting clear (viable) and blue (dead) cells using a Neubauer hemocytometer under phase-contrast microscopy (Motic). Centrifuge the cell suspension at 300 × g at room temperature. Aspirate the supernatant and transfer (a) 15 × 10⁶ cells to fresh LaCell Stromal Medium™ for subculturing. 3 ~150×10 3 Resuspend at a concentration of live cells / ml, then add 1 ml of medium / 5 cm 2(b) Culturing at the surface area density of the flask, or (b) using Mr.Frosty® alcohol bath freezer containers, dispensing into 1.8 ml cryopreservation tubes for immediate cryopreservation in LaCell Cryopreservation Medium®. 6 ~10 7 Resuspend the cells at a concentration of live cells / ml and leave the cryopreservation tubes at -80°C overnight before transferring them to a liquid nitrogen dewar storage container. The quality and characteristics of BM-MSCs can be monitored during the manufacturing process using the following assays.

[0075] Colony-forming unit fibroblast (CFU-F) assay for analysis of adherent cell count and proliferative capacity.

[0076] Lipidogenesis, chondrogenesis, and osteogenic differentiation assays, involving culturing for 14 days in the presence of LaCeli's AdipoQual®, ChondroQual®, or OsteoQual® differentiation induction medium, followed by staining with Oil Red O (Adipoctyes), Alcian Blue (Chondrocytes), or Alizarin Red (Osteoblasts).

[0077] Flow cytometry involving the detection of a panel of surface antigens includes, but is not limited to, the stromal markers CD29, CD44, CD73, CD90, CD105, hematopoietic markers CD11b, CD14, CD45, stem cell marker CD34, mesenchymal marker CD146, and endothelial marker CD31.

[0078] Example 2: Isolation of stromal vascular fraction (SVF) cells and adipose-derived stromal / stem cells (ASCs)

[0079] To isolate SVF cells, first obtain at least 100 ml of liposuction or 50 grams of subcutaneous adipose tissue from a patient undergoing selective liposuction or abdominoplasty. This tissue can be stored at room temperature for up to 24 hours after surgical treatment, which allows for transport or delivery to the laboratory. Upon arrival at the laboratory, transfer the tissue to a sterile BSL2 biological safety cabinet. If the tissue arrives intact (after abdominoplasty), before use, finely chop the tissue into 2-3 mm³ fragments using two sterile or autoclaved scalpels or two pairs of fine scissors. After chopping the tissue or dispensing the aspirated adipose tissue, transfer 100 ml of tissue to a sterile 250 ml capped Nalgene® plastic centrifuge tube. Add an equal volume of sterile PBS and separate the tissue from the liquid phase over 2-5 minutes. Using a 1 ml sterile aspiration pipette, remove the PBS blood layer from beneath the suspended tissue layer. Repeat this procedure 3 to 5 times until the PBS infiltration is no longer blood-colored, but at most pale yellow.

[0080] A PBS solution containing 0.1% collagenase type I (Worthington Biochemical catalog number CLS1, Lakewood NJ) and 1% bovine serum albumin (BSA) fraction V (Fisher Scientific) is prepared by weighing 100 mg of collagenase type I and 1 g of BSA, dissolving them in 100 ml of PBS, and then sterile filtering through a 0.22 micron filter unit in a BSL2 biological safety cabinet. This solution is then warmed to 37°C in a heated water bath or incubator. 100 ml of PBS containing the 0.1% collagenase / 1% BSA solution is mixed with 100 ml of adipose tissue. A 250 ml centrifuge bottle is sealed and placed in a Brunswick shaker incubator at 37°C, and centrifugated at 200–220 rpm for 50–70 minutes. After incubation and agitation are complete, the adipose tissue fragment should show signs of digestion and loss of structural integrity. The digested contents are centrifuged at 300 × g at room temperature for 5 minutes. The resulting pellet is resuspended and the centrifugation step is repeated. The centrifuge tube is returned to a BSL2 biological safety cabinet, and the suspended adipose tissue and supernatant are aspirated. The pelleted SVF cells are resuspended in 15 mL of LaCell Stromal Medium®. A 10 μL aliquot of the cell suspension is removed and mixed with an equal volume of LaCell Live Dead Assay Solution® containing acridine orange / ethidium bromide, and viable (green) and dead (red) nucleated cells are counted using a fluorescence microscope (Motic AE30 Epifluorescent Microscope). Based on the percentage of viable cells and the total number of cells per milliliter, the total number of nucleated cells is calculated using the formula: Total number of viable cells = Percentage of viable cells × Volume (ml) × Total number of cells / ml.

[0081] At this point, the SVF cells are optionally (a) centrifuged at 300 × g for 5 minutes at room temperature, and then 10 6 ~10 7The cells are pelleted by resuspending them in LaCell Cryopreservation Medium (Trademark) at a cell concentration of / ml, dispensed in 1ml volumes into 1.8ml cryopreservation vials, and then cryopreserved overnight at -80°C in a Mr.Frosty (Trademark) container before transferring to a liquid nitrogen dewar for long-term storage. Alternatively, (b) the SVF cell volume isolated from 100ml of liposuction can be resuspended in 105ml of LaCell Stromal Medium (Trademark) to culture and grow to obtain adherent adipose-derived stroma / stem cells (ASCs), and 35ml can be dispensed into each of three T175 flasks. These flasks are incubated in a humidified 5% CO2 incubator at 37°C for 24–72 hours. The degree of cell adhesion is determined by examining the flasks under phase-contrast microscopy at 20x magnification using a Motic microscope. In a BSL2 biological safety cabinet, aspirate the culture medium from the flask before washing the adherent cells twice with 20 ml of sterile PBS preheated to 37°C. Resupply the adherent cell population with 35 ml of fresh LaCell Stromal Medium® or 35 ml of 1:1 fresh :ASC-acclimatized LaCell Stromal Medium® per T175 flask. Maintain the flask in culture until 70–90% confluence is reached, supplying fresh medium or 1:1 fresh :ASC-acclimatized medium every 2–3 days. At this point, the ASCs should be subculturified by digestion with 0.25% trypsin at 37°C in a humidified 5% CO2 incubator. Monitor the adherent cells in the flask by phase-contrast microscopy to confirm that trypsin digestion has released a single-cell suspension. After 5–20 minutes of trypsin digestion, add an equal volume of LaCell Stromal Med™ to stop the enzymatic digestion reaction, and transfer the total volume of cells to a sterile 15 ml or 50 ml conical tube. Remove 10 μl of the cell suspension and mix it with an equal volume of trypan blue solution. Determine the total cell number and the ratio of live cells by counting clear (live) and blue (dead) cells using a Neubauer hemocytometer under a phase-contrast microscope (Motic).This cell suspension is centrifuged at 300 × g at room temperature. The supernatant is aspirated and then (a) transferred to fresh LaCell Stromal Medium (trademark) in 15 × 10⁵ units for subculturing. 3 ~150×10 3 (b) For immediate cryopreservation, the cells can be resuspended at a concentration of live cells / ml and cultured at a density of 5 square centimeters of surface area in 1 ml of medium / flask, or (b) for immediate cryopreservation, in LaCell Cryopreservation Medium (trademark), 10 6 ~10 7 The ASC can be resuspended at a concentration of live cells / ml and dispensed into 1.8 ml cryopreservation tubes using a Mr.Frosty® alcohol bath freezer container. These cryopreservation tubes can then be placed in a liquid nitrogen dewar storage container overnight at -80°C before being transferred to the container. The quality and characteristics of the ASC can be monitored in the process using the following assays.

[0082] Colony-forming unit fibroblast (CFU-F) assay for analysis of adherent cell count and proliferative capacity.

[0083] Lipidogenesis, chondrogenesis, and osteogenic differentiation assays performed by culturing for 14 days in the presence of LaCell's AdipoQual®, ChondroQual®, or OsteoQual® differentiation induction medium, and then detecting the differentiation by staining with Oil Red O (Adipoctyes), Alcian Blue (Chondrocytes), or Alizarin Red (Osteoblasts).

[0084] Flow cytometry involving the detection of a panel of surface antigens includes, but is not limited to, the stromal markers CD29, CD44, CD73, CD90, CD105, hematopoietic markers CD11b, CD14, CD45, stem cell marker CD34, mesenchymal marker CD146, and endothelial marker CD31.

[0085] Example 3: Preparation of an exemplary biological scaffold Another experimental example describes an adipose tissue apparatus containing SVF and silk bioscaffolds. Progenitor cells derived from the stromal vascular fraction (SVF) of white adipose tissue (WAT) form clonal populations and have the ability to differentiate along multiple lineage pathways. However, the literature is hesitant to define adipocyte precursors as "stromal" or "stem" cells. Recent studies have shown phenotypic CD45 - / CD31 - / CD146 - / CD34 + The non-pericyte subpopulation of adipocytes possessing this characteristic has been demonstrated to be mesenchymal, suggesting that it is an endogenous precursor subpopulation within adipose tissue. Frazier et al. demonstrated that stromal vascular fraction (SVF) cells and cultured proliferating adipocytes / stem cells (ASCs) expressing the GFP transgene (GFP-Tg) eccentrically can be fractionated by flow cytometry. Both newly isolated SVFs and cultured ASCs were seeded on 3D silk scaffolds and subcutaneously transplanted into wild-type hosts, with successful single initial transplants and two consecutive transplants (2° transplants). WAT constructs were taken out after 6 weeks, and the presence of GFP-Tg adipocytes and stem cells was evaluated. Flow cytometry, quantitative polymerase chain reaction, and confocal microscopy showed persistence, proliferation, and growth of GFP-Tg cells, respectively. Glycerol secretion and glucose uptake assays revealed that GFP-Tg lipids are metabolically functional. Constructs seeded with GFP-Tg SVF cells or GFP-Tg ASCs showed higher SVF yield from digestive tissue and greater construct weight compared to unseeded controls. CD146 - CD34 + Constructs derived from GFP-Tg ASC populations rich in CD29 are unselected or CD29 + The GFP-Tg ASCs exhibited higher hemoglobin saturation and a higher frequency of GFP-Tg cells compared to their counterparts. These data demonstrate the success of serial transplantation of non-pericytic adipose-derived progenitor cells capable of reconstituting adipose tissue as a solid organ.

[0086] In one embodiment, this disclosure provides an adipose tissue apparatus comprising SVF and decellularized extracellular adipose matrix (ECM). Three methods for optimal adipose tissue decellularization were compared. Method 1 (M1) is an enzyme-based method developed in the laboratory of Dr. Lauren Flynn in Ontario, Canada. Method 2 (M2) is a novel solvent-free method that improves upon the solvent-based method reported by Ji Suk Choi of Hanyang University, Republic of Korea, and Kinam Park of Purdue University. Method 3 (M3) is based on the preparation of a commercially available tumor-derived extracellular matrix product, Matrigel®.

[0087] Human adipose tissue was obtained from informed abdominoplasty patients under an IRB-approved protocol. These studies compared alternative decellularization processes with respect to desired properties in the final ECM product. Bioscaffolds were evaluated for DNA depletion, ECM composition, physical structure, and protein content. Decellularization was confirmed by the absence of cell nuclei present during H&E staining and further confirmed by the absence of residual DNA based on spectroscopic analysis. A wide range of collagen compositions were observed by tricolor staining. Scanning electron microscopy (SEM) revealed complex fibrous physical structures. Mass spectrometry proteomics analysis identified 25 proteins definitively present in the M1 scaffold (with 2+ peptide sequence hits), 143 in M2, and 102 in M3 scaffolds, compared to 155 in native tissue. Several structural proteins were found to be relatively abundant as a result of decellularization, including type VI collagen, fibrillin, and laminin. The results described herein extend to the identification of protein components of adipose tissue-derived bioscaffolds and further define a functional microenvironment that promotes ASC adhesion, proliferation, and differentiation in a solvent-free manner superior to current techniques.

[0088] Experiments with a silk fibroin-based biological scaffold Materials and methods mouse Animal studies were conducted under the supervision of the Department of Comparative Medicine of the Tulane University School of Medicine, under protocols reviewed and approved by the Institutional Animal Care and Use Committee, in accordance with federal, state, and National Institutes of Health policies and regulations. SVF cells and ASCs were isolated from 8- to 12-week-old male C57BL / 6-Tg(UBC-GFP)30cha / J mice (human ubiquitin C promoter-driven green fluorescent protein (GFP) transgene, Jackson Laboratory, Bar Harbor, ME, https: / / www.jax.org) according to published methods. GFP transgene (GFP-Tg) ASCs expressed the GFP transgene upon isolation, maintained in vitro growth for at least 10 passages, and exhibited a cell doubling time of 2-2.5 days. For initial characterization, these cells were examined for the expression of the markers CD11b (Mac-1α; integrin alpha M), CD29 (β1 integrin), CD34 (mucosialin), CD45 (leukocyte common antigen; Ly5), CD90 (Thy-1), and Sca-1 (stem cell antigen 1; Ly6A / E).

[0089] Adipose tissue harvesting and SVF cell preparation

[0090] Following the protocol published by our laboratory, subcutaneous inguinal white adipose tissue (iWAT) was isolated from 8-12 week old male C57BL / 6-Tg(UBC-GFP)30cha / J mice, finely chopped, and digested with collagenase for 60 minutes. In short, these iWAT SVF pellets were collected by centrifugation, washed with phosphate-buffered saline (PBS), filtered through a 70 μm mesh (Millipore, Billerica, MA, http: / / www.emdmillipore.com / ), and the concentration of these SVF cells was determined by automated cell counting (Invitrogen, Carlsbad, CA, http: / / www.thermofisher.com / us / en / home / brands / invitrogen.html). These 1° SVF cells are suspended in Stromal Medium (DMEM / F-12 Ham's, 10% FBS [Hyclone, Logan, UT, http: / / www.hyclone.com], 100U penicillin / 100g streptomycin / 0.25g fubizizone) at a tissue digest density of 0.156 ml per square centimeter of surface area for proliferation and culture to obtain GFP-Tg ASCs, or resuspended at a final concentration of 1 × 10⁶ nucleated cells / ml of PBS in preparation for staining.

[0091] Initial immunophenotype and fractionation of SVF cells Cell suspensions were incubated with antibodies against the cell surface antigens listed in the Supporting Information Table at room temperature (RT) for 30 minutes, protected from light. After washing twice with PBS, flow cytometry analysis was performed using a Beckman-Coulter Galios flow cytometer (BD Biosciences, San Jose, CA, www.bdbiosciences.com). The immunophenotypes and relative subpopulations of these GFP-Tg SVF cells were determined up to passage 2 of plastic-adhesive culture using fluorescent dye-conjugated monoclonal antibodies that detect the following panels of endothelial, hematopoietic, mesenchymal, and stem cell-related antigens, using the scheme shown in the Supporting Information Table.

[0092] Selection of SVF cells Two studies were conducted using GFP-Tg cells derived from GFP-Tg C57BL / 6 mice (Figure 8). These included serial transplantation of unfractionated GFP-Tg SVF cells and serial transplantation of a viable cell-selected and cultured GFP-Tg ASC subpopulation. In the first study, GFP-Tg SVF cells were selected from the GFP-Tg population by flow selection, and the unfractionated GFP-Tg SVF cells were immediately introduced into silk scaffolds for serial transplantation of GFP-Tg SVF cells in non-GFP-Tg mice. In the second study, a GFP-Tg CD146-SVF subpopulation was selected and plated as either (a) an unfractionated control, or (b) CD29-positive, or (c) CD34-positive. These cultured populations (a-c) were classified by immunophenotype and introduced into silk scaffolds for serial GFP-Tg ASC transplantation (ASC serial transplantation study; see the introduction, culture, and transplantation of HexaFluoroIsoPropanol silk scaffolds described later).

[0093] ASC culture and growth Viable GFP-Tg 1° SVF cells were sorted using a BD Biosciences fluorescence-activated cell sorter (FACS) Beckman-Coulter Galios flow cytometer equipped with Galios acquisition software (BD Biosciences, San Jose, CA) featuring two lasers and eight detectors. Compatibility was maintained between the selected fluorescent dyes and the continued use of transgenic GFP for cell sorting purposes. Lineage removal was performed using a cocktail of eFluor-conjugated antibodies detecting mouse (mu)CD3, CD45R, CD116, LyG6, and Ter-119 (eBioscience; 88-7772). Viable SVF cells were selected using APC-conjugated anti-muCD29 (17-0291) and PE-conjugated anti-muCD34 (56-0341). After growing ASCs in plastic-adherent culture, ASCs were isolated using phenotypic and functional criteria established by the International Federation of Adipose Therapeutics (IFATS) and ISCT. In short, 0° (P0) ASCs were trypsinized with 5 ml of 0.25% trypsin (Life Technologies, Grand Isle, NY) for 5 minutes. An equal volume of ASC medium was added to stop the trypsin digestion. P0 ASCs were counted using the trypan blue dye exclusion method and subcultured into new T175 flasks at a density of 0.4 × 10³ cells / cm². These cells were cultured until they reached 70% confluence. These populations were subdivided into (a) an unfractionated control, (b) a population rich in CD29+ (Lin-CD29+ CD146-), or (c) a population rich in CD34+ (Lin-CD34+ CD146-), and cultured and grown up to P2. These (a) unfractionated control, (b) ASC population rich in CD29+, and (c) ASC population rich in CD34+ were individually seeded in flasks and cultured and grown according to the method we have published and validated. After sorting for CD29, 95.03±2% of these cells expressed CD29 and GFP.To obtain a sufficient number of cells for seeding, these SVF subpopulations were immediately plated, grown, and passaged. Note that these cells (CD29+ and CD34+ subpopulations) were selected individually and not sorted based on co-expression of both CD29 and CD34. In addition, the initially sorted CD34 subpopulation (99.85±4% positive for both GFP and CD34) contains other SVF precursor populations that co-express CD34 and are eliminated during the plastic adhesion growth and selection of the adherent ASC population. Therefore, the CD34 expression initially sorted in SVF cells is higher than that of cultured CD34-selected ASCs, which may lose surface marker expression depending on the adhesion and growth process. In passage 2 (P2), aliquots (1 × 10⁵ ASCs) of ASCs derived from each SVF fraction were characterized by analytical flow cytometry for GFP positivity and for the antigens CD29, CD31, CD34, CD45, CD73, CD90, CD105, CD146, and Sca-1, and changes in the profile due to proliferation were monitored. In parallel, live P2 GFP-Tg ASCs were evaluated for in vitro proliferation, colony formation, and lipidogenesis / differentiation, as well as for transplantation studies.

[0094] HexaFluoroIsoPropanol Silk Scaffold Introduction, Culture, and Transplantation HexaFluoroIsoPropanol (HFIP) silk scaffolds, obtained from the Tissue Engineering Resource Center (Tufts University, Medford, MA, http: / / ase.tufts.edu / terc / ), were pre-moistened overnight at 4°C in Stromal Medium (Dulbecco's modified Eagle's-Ham's F-12 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin). These scaffolds were used in studies with unfractionated SVF cells and studies with fractionated passage 2 (P2) ASCs. For SVF studies, newly isolated GFP-Tg iWAT 1°SVF cells were introduced into the opposite side of cylindrical HFIP silk scaffolds in two 50 μl aliquots, each containing a total of 2.5 × 10⁵ cells, using a pipette according to our published method

[33] . For ASC studies, samples of newly isolated GFP-Tg iWAT 1°SVF cells were either plated directly (unfractionated) or sorted based on CD146-CD29+ or CD146- / CD34+ expression. These subfractions were plated and cultured to P2. P2 ASCs derived from the unfractionated SVF control, the CD146-CD29+-rich population, and the CD146-CD34+-rich population were introduced into two 50 μl aliquots, each containing 2.5 × 10⁵ cells, on opposite sides of a cylindrical HFIP silk scaffold using a pipette.

[0095] For both studies, these scaffolds were transferred to a humidified 37°C, 5% CO2 incubator and rotated every 15 minutes for 2 hours. After adding 5 ml of Stromal Medium, each scaffold was immediately transplanted into 8-week-old male non-transgenic C57B1 / 6 mice. A representative set of scaffolds (1° SVF cells or cultured ASC population) was retained, freshly frozen with an optimal cleavage temperature compound (OCT), and stored for use as a positive control. The remaining individual scaffolds introduced with SVF cells or ASCs (n=20 for each subpopulation) were transplanted bilaterally into dorsal subcutaneous pouches prepared for C57B1 / 6 mice according to standard veterinary handling procedures under the approved IACUC protocol (Protocol #4302). Each transplanted mouse was given either an empty scaffold (control), a scaffold seeded with SVF cells, or a randomized combination of an unfractionated control (P2 ASC), a population rich in CD29+ (CD29+ CD146-), or a population rich in CD34+ (CD34+ CD146-).

[0096] Growth assay Immediately after cell seeding, the relative number of metabolically active stem cells in each seeded scaffold was determined by the AlamarBlue® assay according to the manufacturer's instructions. The seeded scaffolds were incubated in Stromal Medium supplemented with 10% Alamar Blue reagent at 5% CO2 and 37°C for 2 hours. Aliquots (100 μl) of culture medium were transferred to a 96-well plate, and the fluorescence intensity was quantified using a microtiter plate reader (Fluostar Optima) with an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Unseeded scaffolds and tissue culture wells were maintained in culture medium as described above and similarly analyzed as blank controls to adjust background fluorescence. The scaffolds were then weighed, and the relative cell numbers were calculated as their relative fluorescence intensity (RFU) per milligram of scaffold wet weight, as described above.

[0097] adipogenic differentiation Culture and growth, and lipid-producing differentiation of scaffold-inoculated non-GFP-Tg ASCs or GFP-Tg ASCs, were carried out over 15 days as described above. In short, cultured ASCs were grown in Stromal Medium (Dulbecco's modified Eagle's-Ham's F-12 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin). The ASCs were then trypsinized and inoculated in ASC culture medium at a density of 3 × 10⁶ per square centimeter. 4Individual cells were plated and allowed to adhere for 24 hours. On day 1 (24 hours after plating), the medium was removed, and the cells were incubated for 3 days in a lipid-producing differentiation medium (Dulbecco's modified Eagle's-Ham's F-12 medium supplemented with 10% fetal bovine serum, 15 mM HEPES [pH 7.4], biotin [33 μM], pantothenate [17 μM, Sigma], human recombinant insulin [100 nM, Boehringer Mannheim], dexamethasone [1 μM], 1-methyl-3-isobutylxanthine [IBMX; 0.25 mM], and rosiglitazone [1 μM]). For the remaining 9 days of the adipocyte differentiation maintenance period, the medium was removed every 3 days and replaced with the same medium (maintenance medium) without IBMX and rosiglitazone.

[0098] Lipid production in scaffold-seeded cells

[0099] For GFP-Tg SVF cells and GFP-Tg ASCs seeded on scaffolds, the scaffolds were autoclaved one day before seeding and immersed in lipid-producing medium at 37°C, 5% CO2, and 95% relative humidity. Subsequently, GFP-Tg ASCs were trypsinized and then, according to our published method, 2.5 × 10¹⁶ cells were placed on the opposite side of the cylindrical HFIP silk scaffolds on the scaffolds that had been pre-soaked in ASC lipid-producing differentiation medium. 5Two 50 μl aliquots containing individual cells were plated using a pipette. These cells were incubated for 3 days in a lipid-producing differentiation medium (Dulbecco's modified Eagle's-Ham's F-12 medium supplemented with 10% fetal bovine serum, 15 mM HEPES [pH 7.4], biotin [33 μM], pantothenate [17 μM, Sigma], human recombinant insulin [100 nM, Boehringer Mannheim], dexamethasone [1 μM], 1-methyl-3-isobutylxanthine [IBMX; 0.25 mM], and rosiglitazone [1 μM]). For the remaining 9 days of the adipocyte differentiation maintenance period, the medium was removed every 3 days and replaced with the same medium (maintenance medium) without IBMX and rosiglitazone. Similarly, control cultures were maintained in parallel in the absence of lipid-producing stimulants. All seeded and unseeded (control) scaffolds were cultured in a humidified incubator at 37°C with 5% CO2. After the in vitro culture period, the seeded scaffolds were evaluated for their degree of lipid production.

[0100] Quantitative determination of cytoplasmic lipids For plastic-adhered cultured ASCs, oil-red-o (ORO) (Sigma-Aldrich) was incorporated into monolayers of GFP-Tg ASCs cultured in adipocyte differentiation medium for 12 days, and lipid formation was evaluated. ORO uptake was quantified. In short, 0.5% (w / v) ORO was prepared in ethanol. Then, 3 parts ORO and 2 parts PBS were mixed to prepare a diluted standard solution. Monolayers of GFP-Tg ASCs cultured in 12-well plates were rinsed three times with PBS, and then fixed with 10% (v / v) formalin (Sigma-Aldrich) for 15 minutes. These monolayers were then rinsed three times with PBS and incubated in the diluted ORO standard solution at room temperature for 45 minutes. After aspirating the unincorporated ORO, the monolayers were rinsed four times with PBS. These stained monolayers were visualized using a phase-contrast microscope (Eclipse 800, Nikon, Tokyo, Japan, http: / / www.nikon.com / ). The stained monolayers were incubated in 100% isopropanol for 10 minutes to extract the incorporated ORO. The absorbance of each aliquot at 510 nm was then measured using a 96-well plate reader.

[0101] For ASCs introduced into the scaffolds, accumulated lipids were also visualized using ORO staining. For histological purposes, these scaffolds were fixed overnight with 2M sucrose. The scaffolds were embedded in OCT medium, frozen with dry ice, and stored at -80°C. The frozen scaffolds were then sectioned (7 μm sections) using a cryostat and stained. For ORO staining, the frozen sections were fixed in 10% buffered neutral formalin for 15 minutes and rinsed with distilled water. A diluted ORO standard solution was placed on the frozen sections and incubated for 20 minutes. The slides were rinsed with distilled water and counterstained with 4',6-diamidino-2-phenylindole (DAPI) according to the manufacturer's protocol. The stained sections were covered with glass coverslips. Images were taken using a phase-contrast microscope.

[0102] Sequential transplantation The transplanted animals were maintained for 6–8 weeks, euthanized, and the scaffolds were removed using sterile dissection techniques. Previous in vivo studies have demonstrated that 4–6 weeks is a sufficient period to enable human ASC lipid production in transplanted silk scaffolds. After 6 weeks, rapid proliferation and differentiation of GFP-Tg cells were observed by confocal microscopy. Therefore, our study was conducted at 6 weeks. For microscopic analysis, a set of n=2 scaffolds was freshly frozen using OCT. The remaining n=18 scaffolds were subjected to collagenase digestion with 0.1% collagenase type I (Worthington Biochemicals, Brunswich, Lakewood, NJ, http: / / www.worthington-biochem.com), 1% bovine serum albumin, and 2 mM CaCl2, with intermittent shaking at 37°C for 60 minutes, followed by 1 hour in supplemented PBS. These 2° (secondary transplant) SVF cells were isolated under HFIP Silk Scaffold as described above.

[0103] Introduction, culture, and transplantation The frequency of GFP-Tg cells in 2°SVF cells, as well as the expression of their associated antigens (the monoclonal antibody (mAb) panel described above), were determined by analytical flow cytometry using samples from each group. The remaining selected 2°SVF cells from the initial cohort were introduced into newly prepared HFIP silk scaffolds by repeating the same process as above. After incubation, these scaffolds were introduced with 2°SVF cells and transplanted bilaterally into C57Bl / 6 mice over a period of 6 weeks. At that point, scaffold collection and analysis were repeated to obtain a 3°SVF cell population. Due to limitations in cell collection, scaffold collection and analysis were completed after the 3°SVF cell population and the 2°ASC population. As a positive control, intact inguinal depots from C57Bl / 6 (UBC-GFP) mice were sequentially transplanted into syngeneic wild-type recipients over the same period. In addition, insulin sensitivity within the extracted silk scaffold constructs was determined using our published in vitro glucose uptake and lipolysis assays to ensure functionality. It should be noted that in this study, we measured the engraftment time between the day of surgical transplantation of the scaffold (with or without cells) and the day of surgical removal from the animal. For the negative control group, tissue was collected from age- and sex-matched non-GFP-Tg C57BL6 mice.

[0104] Hemoglobin saturation measurement The Zenascope PCI spectrometer (Zenalux, Durham NC, http: / / www.zenalux.com) uses a broadband halogen light source (probe) to illuminate target tissue. Zenascope enables the measurement of protein levels in living animals without invasive techniques. A branched optical fiber probe, having a stainless steel jacket at a common stainless steel hard end with a diameter of 0.25 inches and terminating therein, is applied to the tissue surface, delivering light to the target tissue, collecting the reflected light signal, and quantitatively extracting the optical properties of the tissue (wavelength-dependent absorption and scattering coefficients) from the reflectance spectrum measured over the wavelength range of 500–650 nm. From the extracted absorption coefficients, portable, standardized measurement hardware can determine oxyhemoglobin (HbO2), deoxyhemoglobin (dHb), total hemoglobin concentration (Hb, defined as HbO2 + dHb), and hemoglobin Rapid, real-time quantitative analysis of oxygen saturation (sO2, defined as the ratio HbO2 / Hb) is achieved. The optically reduced scattering coefficient is also measured and reported by the instrument, which allows for accurate quantification of tissue chromophore measurements independently of changes in tissue scattering. The integration time was automatically set by the system software for each measurement to maximize the reflectance signal while remaining within the detector's linear response range, and was typically in the range of 100 to 200 ms. The probe's reflectance calibration using a 99% reflectance standard was performed before all experiments and periodically between procedures.

[0105] ImageJ graft analysis To report on ASC or SVF long-term survival time, three representative images of all grafts taken from each cohort were converted to TIF files and exported to ImageJ for analysis. Images of unsown scaffolds (control) were quantified using ImageJ Version 1.46 software (NIH). Pixel values ​​were from the entire tissue graft, not from sectioned images. Thresholds were set by subtracting the number of background pixels using the control. The relative mean number of pixels from the tissue section group was averaged, and the standard deviation was calculated.

[0106] Cryogenic scanning electron microscope After 24 hours of cell culture, scaffolds seeded with GFP-Tg ASCs were subjected to cryo-scanning electron microscopy. Cryo-SEM structural studies were performed using a Gatan 2500 alto Cryo-system and a Hitachi S-4800 scanning electron microscope (SEM). The tissue was cut into 7×5×5 (mm) sections, placed approximately 5 mm above the surface of the cryo-SEM sample holder, fixed and adhered to the holder, and then frozen in liquid nitrogen at approximately -210°C for approximately 30 seconds. This frozen tissue was transferred to a pre-chamber attached to the SEM in a vacuum, then crushed, and sublimated at -95°C for 5 minutes. After coating with Pt / Pd at -130°C for 88 seconds, the sample was transferred to the SEM and observed at -130°C and 3kV.

[0107] histochemistry New frozen tissue samples were sectioned, stained with a lipophilic fluorescent dye (Bodipy), and the co-localization of GFP and lipid signals was evaluated using a fluorescence microscope.

[0108] Quantitative polymerase chain reaction (qPCR) for GFP gene expression Total DNA was isolated from tissue-modified fat pads or positive / negative controls using the Qiagen FFPE tissue prep Mini Kit (Qiagen inc., Valencia, CA, http: / / www.qiagen.com) according to the manufacturer's instructions. Polymerase chain reaction was performed using approximately 500 ng of total DNA with SYBR green real-time PCR DNA binding dye. The reaction mixture was incubated with GFP-specific oligonucleotide primer sets (forward: 5'-TCGCCTACCAGCTCATGCATAACA-3'; reverse: 5'-TGAAGCTCTTCCAGGTGTCAACGA-3') in a real-time thermal detection system (Bio-Rad Laboratories, Hercules, CA, http: / / www.bio-rad.com). All results were normalized compared to glyceraldehyde 3'-phosphate dehydrogenase (GAPDH) expression controls.

[0109] statistics All studies were repeated at least three times, and numerical values ​​were reported as mean ± standard deviation (SD). Individual pairs were compared using Student's t-test, while larger groups were analyzed using analysis of variance. Findings were defined as significant if the minimum p-value was ≤.05.

[0110] result Isolation and characterization of the cells used Figure 1A shows a representative subpopulation of cells detected in GFP-SVF of iWAT isolated from male mice aged 8–12 weeks. The initial SVF contained an average of 3–5 × 10⁶ cells per milliliter of adipose tissue. 5It produced 100% 1° (primary) SVF cells, which is close to the figures reported in the literature. Based on surface immunophenotype, the three largest subpopulations were preadipocytes (24.7±6.9%; CD14-, CD36+), ASC-like cells (20±10.7%; CD146-, CD34+, CD90+), and leukocytes (19.4±9.7%; CD34-, CD45+). Notably, this pelletized SVF cell population excludes floating mature adipocytes (estimated to be about 30% of the total population) due to the process of collagenase type I tissue digestion and isolation by differentiation centrifugation. The initial SVF fraction was first sorted for the CD146- subpopulation (80.5±5%). This served as an initial cohort for the fractionated SVF cells. Other cohorts were selected based on CD34 and CD29 surface antigen expression, i.e., (b) CD29+ (18.2±8.5% of the CD146- population) and (c) CD34+ (21.4±12.8% of the CD146- population) SVF cells. The flow cytometry sorting protocol is shown in Figure 2. The characteristics of these cohorts [(a) all CD146-SVF cells, (b) cells rich in CD146- and CD29+, and (c) cells rich in CD146- and CD34+] were determined based on immunophenotype, proliferation, colony formation, and potential for lipid formation in plastic-adhered cell cultures and passage 2 (P2). Similar to other published reports, interstitial marker (CD29) expression was elevated, while endothelial markers (CD31, CD45) and hematopoietic markers (CD34, CD11b) decreased with passage. Sca-1 expression was 61.6±10.7% on average in the unselected SVF population and increased throughout passage (Figure 2A). Enrichment of CD29 and CD34 reduced Sca-1 expression (CD29: 13.8±2.4%, CD34: 13.7±0.9%). Subpopulations rich in CD34+ showed significantly higher proliferative and lipid-producing abilities than subpopulations rich in CD29+ (Figure 2B). Furthermore, the CD29+ subpopulation had a slower doubling time and less intracytoplasmic lipid accumulation than the unselected population (Figures 2C, 2D).

[0111] Seeding of SVF and ASC onto scaffolds The in vitro seeding and proliferation characteristics of GFP-ASCs were investigated after seeding on HFIP-based silk scaffolds. Confocal microscopy was performed on seeded and unseeded control scaffolds 24 hours after incubation (Figure 9). GFP detection and Alamar Blue staining indicated the priority of cell localization at the ends of the silk scaffold honeycomb-like structure (Figure 9B, bright-field, and Figure 9D, scanning electron microscopy). Cells remaining at these ends began to differentiate (Figure 9C) and proliferate (Figures 9D, 9E). Alamar Blue (Figure 9D) cytosol and DAPI (Figure 9E) nuclear positivity quantification showed that cells were present 2 hours after seeding (18 hours; 16±2 cells; 2 hours, 11±8 cells; Figure 9E), but they were attached to the scaffold and showed metabolic activity measurable by RFU 18 hours after seeding (18 hours, 36 RFU; 2 hours, 0.001 RFU; Figure 9E).

[0112] SVF cells enhance engraftment and generate fat in vivo. Unfractionated SVF cells were monitored for proliferation and lipid synthesis over a 6-week period. Photographic analysis revealed that the presence of SVF cells promoted engraftment by week 1 (Figure 3A). Percent hemoglobin saturation (%Hb; Figure 3B) also supported the observed promotion of SVF cell-mediated angiogenesis. It should be noted that hemoglobin measurements were performed on the skin surface of living mice, and saturation measurements evaluate all tissues, not just arterial blood supply. From week 2 onward, no significant difference in engraftment was observed visually or quantitatively using ImageJ of explant images (Figure 3C). This is consistent with published data suggesting vascular and supportive properties of the scaffold that promote lipid synthesis in vivo. Scaffold mass (Figure 3D) and total SVF recovered from seeded scaffolds (Figure 3E) were significantly larger than unseeded controls during week 1. SVF cell counts and scaffold mass reached their peak in week 2 and then decreased by week 4 (Panels 3D, 3E).

[0113] Unselected GFP-SVF cells detected after serial transplantation Weekly detection of unselected GFP-SVF cells in initial transplants from week 1 to week 6 (Figure 4A) revealed that GFP-SVF cells not only persist within the scaffold but also proliferate and differentiate as early as week 1 after transplantation. Confocal microscopy images of the initial SVF explant at weeks 1, 4, 5, and 6 are shown (Figure 4A). GFP-Tg SVF cells were detected in explants derived from the initial cohort (T0 transplants), as well as from initial transplants (T1 transplants) and secondary successive transplants (T2 transplants; Figure 4B). Flow cytometry-based detection of GFP-Tg SVF cells was consistent with confocal microscopy observations (Figure 4C). In the initial transplants, 19% were GFP antigen positivity via flow cytometry at week 4, while in T1 and T2 transplants, GFP positivity was observed at 16.2% and 13.1%, respectively, at the same time point compared to the initial transplants (Figure 4C). GFP quantification, BODIPY lipophilic dye staining (to demonstrate the lipidogenesis of the construct), and co-localization of nuclear DAPI staining were performed using ImageJ. Quantification showed a 50% increase in the percentage of GFP-Tg SVF cells by 6 weeks after T0 transplantation compared to 1 week (Figure 4D). It is important to note that the flow cytometry data reported in Figure 4D were performed on SVF cells isolated from the tissue reported in Figure 4C. Adipose tissue contains mature adipocytes and other cells that cannot withstand the tissue digestion procedure and are not reflected in the flow cytometry count. Therefore, Figure 4D does not reflect all cells in the tissue sample. GFP expression in the isolated explants was further investigated based on protein and DNA levels via flow cytometry and qPCR, respectively (Figures 4E, 4F). Weekly detection of 10% GFP-Tg cells via flow cytometry revealed an increase in expression from 3.8% at 2 weeks post-T0 transplantation to 16% at 4 weeks (Figure 4E). After T2 transplantation, GFP DNA expression was enriched by more than 200 times (unseeded scaffold transplantation in non-transgenic mice) (Figure 4F). Surface immunophenotypes of cells from the T2 transplantation population were also compared with GFP-Tg controls and unseeded scaffold controls after 6 weeks (Figure 4G).This T2 scaffold showed 36.3±2.4% GFP positivity, 67.8±4.1% CD29 positivity, and 52.4±2.4% CD45 positivity (Figure 4G).

[0114] GFP-Tg ASCs rich in CD34 enhance engraftment and generate functional fat in vivo. GFP-Tg SVF cells were isolated and classified either as unfractionated cells or as groups rich in CD146-CD29+ or CD146-CD34+. These cells were cultured and grown to P2 GFP-Tg ASCs, seeded on silk scaffolds, and transplanted into non-transgenic mice up to T0 serial transplantation. At 6 weeks after graft removal, no visual difference in fat depot size was observed (Figures 5A, 5B). Percent hemoglobin (%Hb) saturation and explant engraftment were measured. Grafts seeded with both CD29- and CD34-rich GFP-Tg ASCs showed higher %Hb saturation 1 week after transplantation compared to unseeded grafts and grafts seeded with unselected ASC controls (Figure 5C). Furthermore, grafts rich in CD34- showed significantly higher %Hb saturation than grafts rich in CD29- (unsown ASC: control, 37.6±9.9%; CD34-rich, 68.1±5.3%; CD29-rich, 45±10.8%; Figure 5C). This was confirmed visually and quantitatively using ImageJ software analysis of microscopic images (Figure 5D). Measurement of explant mass and peripheral fat mass (Figure 5E) revealed that artificial constructs derived from CD34-rich grafts were significantly denser than CD29-rich, unsorted GFP-Tg ASCs, and unsown controls (Figure 5E). Total SVF recovered from scaffolds inoculated with CD34-rich ASCs (Figure 5F) was also significantly higher during week 1 than that of unselected CD29-rich ASCs and uninoculated controls (Figure 5F). Further studies of the functionality of the tissue-modified fats revealed that the CD34-rich constructs secreted glycerol at levels comparable to those of unselected ASC constructs, while the CD29-rich constructs secreted significantly lower levels of glycerol (Figure 5G). No significant differences in glucose uptake were observed between cohorts (Figure 5H).

[0115] CD146-CD34+ / CD29+ GFP-Tg ASC detected after serial transplantation GFP-Tg ASCs were detected within 6 weeks of the initial cohort (T0 transplants) and the initial continuation (T1 transplants; Figure 6A). These tissue grafts were infiltrated with blood vessels, which is consistent with the early observation of tissue engraftment using scaffolds seeded with GFP-Tg SVFs and GFP-Tg ASCs. Flow cytometry-based detection of newly isolated GFP-Tg cells supported GFP positivity on confocal microscopy observation 6 weeks after transplantation (Figures 6C, 6D). Flow cytometry revealed GFP antigen positivity in 29.4% of the initial CD34--rich construct compared to 10.9% of the CD29--rich construct, which showed GFP positivity (Figure 6C). CD34--rich and CD29--rich T1 grafts showed GFP positivity in 20.4% and 8.4% of the constructs, respectively (Figure 6D). GFP, BODIPY lipophilic dye, and DAPI co-localization staining were quantified using ImageJ. GFP expression in explants was further investigated based on DNA and protein levels via qPCR and flow cytometry, respectively (Figures 6E-6G). Detected GFP DNA was 15-fold higher in the CD34--rich construct compared to the CD29--rich construct, and more than 10-fold higher in the unselected ASC construct after T1 transplantation (Figure 6E). Percent GFP-Tg cell detection via flow cytometry supported a significant difference in GFP-Tg cell expression from CD34-rich constructs compared to CD29-rich constructs after 8 weeks, for T0 grafts (CD29, 9.8±1.1; CD34, 24.7±6.5; Figure 6F) and T1 grafts (CD29, 5.4±1.2; CD34, 17.5±1.4; Figure 6G).

[0116] Constructs rich in CD146-CD34+ produce functional GFP+ fat in vivo. We extracted 8-week consecutive grafts from seeded GFP-Tg SVF, unsorted GFP-Tg ASC, GFP-Tg CD34--rich ASC, GFP-Tg CD29--rich ASC, and GFP-positive and negative controls, and analyzed their GFP positivity (Figures 7A-7C) and functionality (Figures 7D, 7E). Confocal microscopy images of 8-week perifat structures extracted after BODIPY staining revealed a significant increase in the percentage of GFP-Tg adipocytes in the CD34--rich cohort compared to unsorted ASC and SVF-seeded structures rich in CD29- (Figures 7A, 7B). Image quantification using ImageJ was consistent with visual observation (SVF, 2.5±1.7%; unselected GFP-Tg ASC, 15.5±0.2%; CD29-rich, 6.1±7.0%; CD34-rich, 31.3±0.4%; Figure 7C). Further studies of tissue-modified adipose function showed that the CD34-rich construct secreted glycerol levels comparable to the unselected ASC construct, while the CD29-rich construct secreted significantly lower levels of glycerol (CD34, 70±16.1μM; unselected ASC, 75±22.1μM; CD29, 40±14.6μM; Figure 7D). Similar to the constructs isolated in Figure 5, no significant differences in glucose uptake were observed between cohorts (Figure 7E).

[0117] Consideration This study successfully transplanted newly isolated GFP-Tg SVF cells into non-GFP-Tg transgenic syngeneic mouse hosts using a 3D silk matrix in a single initial transplant and two consecutive transplants (2° transplants). Initial time-based graft removal studies demonstrated the survival, proliferation, and differentiation of GFP-Tg SVF cells, leading to the formation of metabolically functional adipose tissue. Time-based GFP-Tg SVF experiments also revealed the ability of SVF cells to enhance engraftment and shorten engraftment time. Proliferated plastic-adherent CD146-CD29+ and CD146-CD34+ P2 GFP-Tg ASC populations were seeded in a 3D matrix and successfully transplanted into non-GFP-Tg transgenic hosts in initial and consecutive transplants (1° transplants). To our knowledge, we are the first to report the ability of a specific subpopulation of non-pericytic ASCs within interstitial vascular compartments to generate functional adipose tissue through serial transplantation.

[0118] Mauney et al. reported on the ability of cultured, cryopreserved ASCs, which were not selected or enriched by flow cytometry, to generate fat pads in rats using modified scaffolds. An objective of their study was to compare silk fibroin-derived biomaterials prepared by aqueous (AB) and organic (HFIP) solvent-based processes with collagen and polylactic acid-based scaffolds for their usefulness in adipose tissue modification strategies. In contrast, this disclosure provides 1) a distinct, more heterogeneous stromal vascular population, such as surface antigen-selective subpopulations and other cells within SVFs, which more effectively promote and retain lipid synthesis and adipose engraftment; 2) serial transplantation as a model to illustrate the behavior of cells in this disclosure; and 3) long-term in vitro culture.

[0119] Due to the limited volume of mouse tissue, the ASCs used in this study were pooled from multiple mouse donors, and the initial sample size for serial transplantation was n=20. A key consideration in this study was the use of CD34- and CD29--rich ASC populations grown from newly isolated mouse GFP-SVF, rather than populations of CD34- or CD29- alone. Due to the rarity of CD29+ / CD34+ double-positive and CD29- / CD34- double-negative subpopulations within the newly isolated SVF subfraction, the classification in this study included cultured and grown CD34+ cells without considering CD29 co-expression. The same selection criteria were applied to CD29 cells without considering CD34 co-expression. Therefore, subpopulations within the CD34+ GFF-ASC population also co-expressed CD29, which is involved in fixation, proliferation, migration, and lipidogenesis regulation, as well as other cell surface and intracellular stromal markers. The CD34+ population in newly isolated mouse SVF cells averaged 21.4% in the unclassified population, but the CD34+-rich population in these SVFs maintained an average positivity rate of 33% after two passaging cycles. The percentage of CD34+ GFP-Tg ASCs in this study is consistent with previously published reports on CD34 positivity in human SVFs. This human CD34-positive population demonstrated enhanced proliferative and lipid-producing capabilities in vitro. A strong correlation was also identified between CD34+ ASC yield and the retention of xenograft adipose tissue volume, suggesting that the concentration of CD34+ precursors can predict the survival of human adipose grafts in clinical settings. Future experiments will develop this study into a more humanized system, first by investigating the behavior of selected human cells in an immunodeficient mouse model. These experiments will also allow for larger initial sample sizes, exceeding n=20.

[0120] Current literature uses the terms “adipocyte stroma” cells and “perivascular stem” cells interchangeably to refer to cells that support fat graft survival. Traditionally, perivascular stem cells are identified based on the immunophenotypes CD45-, CD146+, and CD34-. Results from serial ASC grafts rich in CD146-CD34+ indicate the presence of a non-pericyte ASC subpopulation within SVF that can generate a functional fat pad in mice across serial grafts. These findings are supported by in vivo cytoregradation / mapping studies labeling endogenous stem cells in neonatal mice with bromodeoxyuridine (BrdU). Eight weeks later, histological analysis of inguinal fat depots revealed BrdU-labeled stem cells near mature adipocytes (corresponding to the non-pericyte population), in the perivascular space (corresponding to the pericyte subpopulation), and in locations separated from capillaries. In this study, the CD146- population constituted the majority (80%) of the plastic-adhering population, but only 20% of these cells co-expressed the progenitor cell antigen CD34. Nevertheless, the current results indicate that non-pericytes can serve as lipid-producing stem cells in vivo.

[0121] Confocal fluorescence imaging of %Hb saturation at weeks 1 and 4 demonstrated that the presence of GFP-Tg SVF, or richly enriched GFP-Tg ASC, promoted scaffold engraftment as early as week 1 after transplantation. Measurements of SVF obtained from explants and scaffold weight at weeks 1 and 2 supported these data. Published reports suggest that SVF and ASC promote enhanced engraftment, accelerated wound healing, and improved pathophysiological conditions in a variety of applications, including both soft and hard tissue remodeling

[42] . Mesimaki et al. demonstrated the use of proliferated ASC for strengthening maxillary tissue fragments with β-tricalcium phosphate and bone morphogenetic protein 2 (BMP2). The addition of proliferated ASC aided in the development of mature bone structure and vascular system. Yoshimura et al. reported high surgeon and patient satisfaction in 55 patients who underwent a procedure called "Cell-Added Fat Grafting (CAL)," which involves the introduction of SVF cell-reinforced adipose tissue grafts instead of breast augmentation, 12 months post-surgery. More recently, Bura et al. have used autologous human ASCs to treat patients with severe ischemic limbs and have observed a significant improvement in tissue oxygen delivery. Taken together, these human clinical trials lend credibility to our findings in our mouse preclinical model.

[0122] Confocal microscopy and flow cytometry analysis of grafts over eight consecutive weeks demonstrated that a subpopulation rich in CD34+- could generate functional fat pads, similar to unsorted ASC grafts. CD34+- rich ASCs proliferated and underwent lipid-producing differentiation at a higher rate in vitro than CD29+- rich and unsorted ASCs. In vivo, a higher proportion of SVF cells were recovered from the CD34- rich construct. These explants had, on average, higher scaffold mass than CD29- rich and unsorted ASC cohorts. Functional analysis of peri-scaffold fat at six weeks clearly showed that CD34- rich explants secreted more glycerol than CD29- rich, unsorted ASCs and unseeded control grafts. GFP-Tg SVF and -ASC were detected in the surrounding fat depot. Bodily staining, glycerol uptake, and lipolysis analysis suggest that these cells proliferate, differentiate within the scaffold, and migrate locally as mature adipocytes or preadipocytes. These data support CD146 - CD34 + This suggests that subpopulations phenotypically found within the ASC population, which is rich in CD34, generate functional fat pads over successive passages. These findings are consistent with the study by Philips et al., which found that CD34 in donor adipose tissue is present in the tissue. + We clarified the functionality and volume maintenance of human adipose tissue xenografts, which correlate with cell frequency.

[0123] conclusion Distinguishing between "stromal / precursor" cells and "stem" cells remains challenging. Nevertheless, current serial transplantation findings suggest that adult mouse inguinal WAT contains a selectable subpopulation of cells capable of contributing to flow cytometry, functional adipose tissue depot regeneration, and serial transplantation. These findings confirm and build upon the earlier research by Rodeheffer et al., which investigated the regenerative capacity of flow cytometry-selected adipocytes for adipose depots in lipodystrophy mice. In addition, these further validate the usefulness of autologous adipocytes for functional tissue modification. This study also builds upon previous research characterizing the immunophenotype and pericyte properties of isolated adipocyte progenitor cells.

[0124] Example 3: Experiment on the preparation of human platelet lysate (hPL) gel When hPL and DMEM F12 are combined and heated at 37%, the resulting product is a thermoreactive bioscaffold that can be grown (1-2 months) as a 3D culture and / or for in vivo transplantation via subcutaneous injection.

[0125] method hPL preparation Unless otherwise specified, all procedures should be performed in a biological safety cabinet. Collect the desired amount of concentrated, expired human platelets into 15 mL or 50 mL conical tubes. If not to be used immediately for 3D cell culture, store at -20°C for three freeze / thaw cycles before preparing the lysate. Lyse the platelets by osmosis by performing three freeze / thaw cycles. This releases growth factors for cell culture in maximum yield. Centrifuge at 8,000 × g for 20 minutes. Remove the solid (white) upper layer of human platelets by aspiration.

[0126] hPL supplementation medium preparation Prepare hPL supplementation medium by adding 7.5% (v / v) hPL to 1% antibiotic / antifungal agent containing nutrients, amino acids, and growth factors necessary for cell culture, and 91.5% Dulbecco's Modified Essential Medium or other relevant cell culture medium. Do not warm the mixture in a water bath after mixing. Maintain at room temperature.

[0127] Activation of the hPL Matrix Device The prepared hPL is combined with a cell population or a mixture of cell populations to activate gelation (formation of a thick matrix) that promotes cell secretion, proliferation, and differentiation of the extracellular matrix. After mixing these cells with hPL, the plate is placed in an incubator for 20 minutes to activate gelation. Proceed to proliferation, differentiation, transplantation into mice, or other measurement endpoints.

[0128] Example 4: Fat Technology on a Chip Excluding skin cancer, breast cancer is the most common tumor among women in the United States and remains the second leading cause of cancer-related death. The risk of breast cancer death is particularly high among African American women, and the disease is often associated with a poor prognosis due to factors related to diet / nutrition, economic disparities, genetics, access to health, and obesity. Patient-derived xenograft (PDX) models maintained in immunodeficient mice are promising as preclinical models for developing novel breast cancer therapies, but the number of PDX breast cancers commercially available for contract research and oncological pharmacological development is limited. The inventors, a minority, female-owned biotechnology company, are uniquely located in the metropolitan area of ​​New Orleans and the neighboring southeastern Louisiana, providing access to breast tumor specimens from an ethnically diverse patient demographic.

[0129] The inventors have attempted to construct specific information regarding the following: (1) Human platelet lysates contain high concentrations of growth factors that enhance angiogenesis and have been reported to promote colony formation by primary breast tumor cells; (2) Stromal vascular fraction (SVF) cells isolated from subcutaneous adipose tissue contain lymphocytes, myelocytes, pericytes, endothelial progenitor cells, and preadipocytes necessary for developing three-dimensional adipose depots; and (3) Human adipose-derived stromal / stem cells (ASCs) promote the proliferation, migration, and invasion of breast cancer in vitro and in vivo through the secretion of adipokines (particularly leptin) and related mechanisms. Decades of research using established human breast cancer cell lines have produced thousands of manuscripts, but these two-dimensional culture systems often fail to accurately mimic the macroenvironment of breast cancer in terms of immune and stromal cell interactions. Only a relatively limited number of studies using patient-derived xenografts (PDXs) are beginning to address the still unsatisfactory need for pathophysiologically accurate and reproducible human disease models. Much of this research is hindered by the long time required to establish human xenografts in mice and their slow engraftment rates. We have developed an innovative method to address these limitations and needs.

[0130] Human SVF cells isolated from subcutaneous adipose tissue liposuction were cryopreserved and quality-controlled based on flow cytometry characterization, colony formation, proliferation, and differentiation into lipidogenesis and osteogenicity (data not shown). The behavior of cryopreserved SVF cells was evaluated in a 3D mammalian platelet lysate culture system (Figures 16 and 17). These hSVF cells showed increased proliferative capacity in the dose-dependent presence of mammalian platelet lysate compared to a control medium containing 10% FBS (lot characterization and screening based on optimal adipose-derived stroma / stem cell proliferation and lipidogenesis). Furthermore, these hSVF cells increased their spheroid formation in the presence of gradually increasing concentrations of mammalian platelet lysate (Figure 16). In the absence of lipidogenesis-inducing factors, hSVF cells in mammalian platelet lysate containing scaffolds showed vascular-like network formation over a 3-week culture period. In the presence of the added lipid synthesis inducer, these hSVF cells accumulate lipid vacuoles and become detectable by microscopic observation and Bodipy staining (Figure 17).

[0131] Functionality was verified by quantitative biochemical evaluation of adipokine (adiponectin, leptin) secretion in white (WAT) adipose tissue-modified constructs. Adipokine secretion was stronger in the 3D construct compared to the 2D construct (data not shown). Functionality was further verified by quantitative physiological evaluation of lipolysis, glucose uptake, and cellular heterogeneity based on flow cytometry (data not shown).

[0132] This hSVF cell / mammalian platelet lysate scaffold is used in immunodeficiency (nude). nu / nu The substance was transplanted into mice to create a physiologically humanized fat pad (Figure 18). These preliminary data demonstrate the inventors' technical expertise in developing novel, functional, and commercially viable fat chips.

[0133] Adipose-derived cells promote the proliferation of breast cancer cell lines: Research by PI and her collaborators has demonstrated that human adipose-derived cells promote the proliferation of breast cancer cell lines in vitro and in vivo. The obesity index of adipose tissue donors contributes to the proliferation of breast cancer cells. Obesity, in contrast to leanness, enhances breast cancer cell proliferation in donor-derived stromal cells through their increased secretion of adipokin leptin. Therefore, by promoting lipid production of hSVF cells within humanized "fat on a chip," mammalian platelet lysates directly influence the engraftment and proliferation of breast tumor xenografts in immunodeficient mice.

[0134] Statistics: All experiments in Experiments 1 and 2 were performed on triplicate animals, each with two grafts. Quantitative results are evaluated and reported as mean ± standard deviation. Comparison between the control (SVF cells or tumor graft without a scaffold) and the experimental conditions (SVF cells and / or tumor graft with a scaffold) is evaluated based on a two-sided t-test, ANOVA, or Tukey's test (95% confidence interval, p-value < 0.05 is considered significant).

[0135] Hypothesis 1 and Experiment 1: Human platelet lysates function as a scaffold for the proliferation of breast cancer cell lines transplanted into immunodeficient mice (NSGs) in combination with human SVF cells, and function at an equal or better level than Matrigel®.

[0136] Rationale: The oncology community has considerable interest in expanding the scope of PDX models for prognosis, diagnosis, and treatment purposes, particularly in the context of health disparities faced by African American patients. The main challenges are (a) that 40–80% of PDXs fail to successfully transplant into immunodeficient hosts, and (b) the lack of sensitive non-terminal methods for monitoring transplant success or failure in real time [10, 20]. Studies of PDX models of colon cancer have demonstrated that including Matrigel® with xenografts of primary tumors significantly increases the rate of successful engraftment after transplantation into immunodeficient mice. Although the exact mechanism of Matrigel is still unclear, its ability to increase and accelerate tumor growth rates is attributed to the contribution of growth factors and cytokines to the tumor microenvironment. This is consistent with the fact that Matrigel® can promote breast cancer cell proliferation in vitro. The inventors' patented products (mammalian platelet lysates and adipose-derived SVF cells) possess unique characteristics as sources of cytokines and growth factors, and furthermore, when incubated at body temperature, they can create a three-dimensional cellular matrix. In addition, Innogenomic's highly sensitive human cell detection method enables early detection and quantitative evaluation of tumor cell proliferation in living animals without requiring host euthanasia. Experiment 1 establishes proof of principle data demonstrating the capabilities of adipocytes and / or the matrix using one or more human breast cancer cell lines to accelerate tumor growth in vivo.

[0137] Experimental design: Strictness and Transparency: Immunodeficient mouse strains undergo rigorous analysis and quality assurance and are obtained directly from the Jackson Laboratory, an NIH-supported contract research facility. Established breast cancer cell lines undergo rigorous analysis and are supplied directly from the American Type Culture Collection, an NIH-supported cell storage facility. The inventors maintain key demographics of SVF cell donors (age, sex, body mass index, ethnicity) in anonymous records. All cryopreserved vials of the company's cell products are entered into up-to-date databases, and the integrity of storage containers is monitored daily by the company's technical staff. The inventors routinely perform quality assurance and quality control testing on their mammalian platelet lysates and SVF cells using in vitro culture, flow cytometry, and differentiation assays. Because the incidence of breast cancer in women is almost two orders of magnitude higher than in men, Phase I trials are conducted using female NSG mice and hSVF cells isolated from female donors. Other studies involving male mice and male fat donors will be considered in Phase II only if recommended in the studies section.

[0138] Methods: All studies will be conducted under appropriately reviewed and approved IRB protocols (Western IRB or Ochsner IRB), with patient consent (signed or waived), and under the IACUC protocol (Tulane University IACUC). Isolation and Characterization of SVF Cells: Human SVF cells will be isolated from subcutaneous adipose tissue provided by healthy subjects undergoing selective liposuction, under the protocol of LaCell LLC, the inventor's science partner. The tissue will be washed with PBS, digested with type 1 coilagenase, and then the SVF cells will be isolated by density centrifugation and cryopreserved as described above. Initial studies will use pooled hSVF cells from obese female donors with a BMI > 30, n=3. Further studies will be conducted using pooled hSVF cells from lean female donors with a BMI < 25, n=3, where time and resources permit. Upon thawing, the viability of SVF cells will be assessed using a fluorescence-based viability / death assay (LaCell LLC reagent).

[0139] Lipid production in hSVF cells / scaffolds: These hSVF cells (10 6 The SVF was mixed with 25% Matrigel or mammalian platelet lysate in 250 μl of each well of a 12-well plate and cultured in Stromal Medium (LaCell LLC) for 1 week. The SVF / scaffold medium was then replaced with AdipoQual (LaCell LLC) for another week, and "fat on the tip" was monitored with a light microscope to visualize lipid vacuolar accumulation.

[0140] Breast cancer cell lines in in vivo transplantation: Human breast cancer cell lines MCF7, ZR75, and / or T47D are obtained directly from the American Type Culture Collection and grown in vitro. Female NSG mice are anesthetized, a midline incision is made with a scalpel, and then the bilateral sacral cavities are subcutaneously incised from the dorsal flank by blunt incision. 106 breast cancer cells are transplanted into the bilateral dorsal flanks of immunodeficient female NSG mice before being treated with cells alone, cells containing only Matrigel or mammalian platelet lysates, or 10 cells as outlined in the table for Studies 1 and 2[6]. 6 One obese or lean hSVF cell is added. The tumor growth rate is assessed every two weeks based on caliper measurements of the tumor(s) width and length, and serum detection of circulating human genomic DNA Alu. If the tumor volume exceeds 20 mm in diameter, the animals are euthanized by CO2 asphyxiation and cervical dislocation according to a protocol approved by IACUC. [Table 1]

[0141] Histology and immunohistochemistry: The tumor is fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) as described above [17–19]. The tumor sections are stained with antibodies that detect vascular (CD31), bone marrow (GD11b, or CD14), lymphocyte (CD3, CD19), pericytes (CD146), or stromal (CD44, or CD73) surface antigens, using an appropriate secondary antibody conjugated to alkaline phosphatase or horseradish peroxidase for detection. Image analysis is performed using CellProfiler software (http: / / cellprofiler.org / ).

[0142] SA1: Expected Results: In Study 1, the inventors expect to establish tumors with a diameter of 20 mm after 5 weeks following transplantation of MCF7 cells. The addition of Matrigel alone is expected to accelerate the growth rate of MCF7 tumors, comparable to or exceeding that of mammalian platelet lysates alone. The histology of these tumors is relatively homogeneous and dependent on mouse host cells for any stromal or vascular networks. In contrast, the presence of obese hSVF cells in any matrix leads to a further acceleration of tumor growth due to the presence of progenitor cells that can provide stromal and vascular networks, the latter being histologically evident by increased cytotoxicity based on H&E and immunohistochemical staining. The maximum growth rate is achieved by adding pre-conditioned hSVF cells and scaffolds under lipid-producing conditions. This is thought to be due to increased leptin production and secretion, which is believed to be related to the mechanism by which obesity contributes to breast cancer growth. In Study 2, the inventors believe that obese SVF cells promote more rapid tumor growth in all breast cancer lines compared to lean SVF cells, due to their increased leptin secretion. Mammalian platelet lysates are thought to promote cell line proliferation equivalent to or greater than that observed with Matrigel.

[0143] Potential oversights and alternative methods: While previous studies have used cultured ASCs, the inventors believe that SVF cells function equally or even better than ASCs due to their heterogeneity and inclusion of endothelial progenitor cells with angiogenic potential. Nevertheless, it is possible that SVF cells do not steadily accelerate tumor cell proliferation in the same way as ASCs. If this is observed in Study 1, conduct another study with a higher SVF cell:MCF7 cell ratio (3.3 or 10:1). Add an additional control using ASCs instead of SVF cells to the study design. Furthermore, based on previous studies using ASCs, the inventors believe that obese SVF cells steadily enhance tumor growth more effectively than lean SVF cells. This issue concerns the mechanism of stromal / scaffold / tumor interaction and therefore goes beyond the initial intention of "proof of principle" in this study. As a result, the inventors will design Study 2 to compare lean vs. obese hSVF cells using three different established tumor cell lines, provided budget, schedule, and human resources allow. By then, the inventors will have successfully developed PDX tumors induced "in-house," so this delay may prove advantageous. The company's proprietary PDX model can then be evaluated in parallel with the established tumor cell lines to examine the relative contributions of lean vs. obese SVF cells to the PDX tumor growth rate.

[0144] Experiment 1 and Hypothesis 1: Mammalian platelet lysates function as an in vivos cafold, equivalent to or better than Matrigel®, for establishing xenografts derived from breast cancer patients when transplanted into immunodeficient mice (NSGs) in combination with human SVF cells.

[0145] Rationale: While Matrigel is routinely used in in vitro cell culture of breast cancer cell lines, it is not routinely reported as a component in establishing and maintaining PDX models. Nevertheless, the colon cancer literature shows that Matrigel improves and promotes PDX engraftment and subsequent tumor growth. Although the improvement of colony formation from primary breast cancer by platelet lysates was first reported 30 years ago, there has been little research using platelet lysates in breast cancer cell culture or PDX models since then. The inventors have developed a novel, proprietary product that combines adipose-derived cells with human platelet lysates to create a "fat on a chip" model. Since adipose tissue and obesity promote breast cancer growth, the inventors utilize this technology to accelerate and enhance breast cancer PDX growth in immunodeficient mouse models. A commercially available human Alu-based detection assay is used. The inventors can continuously and effectively monitor breast cancer PDX engraftment and growth in individual mice in real time without sacrificing experimental animals. This advanced assay reduces the manufacturing cost of PDX models and accelerates their commercialization.

[0146] Experimental design: The certification of biological reagents will be carried out as outlined in Experiment 1. In addition, the inventors will obtain tumor specimens from a cooperating hospital-based biobank under an approved IRB protocol, with the signed consent of the patient, or, if it satisfies the newly revised FDA regulations and is approved, with signed consent that includes a waiver of rights. All specimens provided to the inventors will remain anonymized in terms of specific information. The inventors will only receive patient information relevant to the tumor and the patient's demographics (age, sex, body mass index, ethnicity, medical history, social history, and tumor histology). Only the biobank and the investigators designated under its IRB protocol will have access to additional information about the tissue donor.

[0147] Methods - The technical procedures will be carried out as described in Experiment 1. The tumor specimen will be aseptically cut into 3 mm³ fragments and transplanted subcutaneously into both flanks of anesthetized NSG mice. The tissue fragments will be transplanted in or out of 250 μl of Matrigel or mammalian platelet lysates, and in or out of 10⁶ SVF cells. The scaffolds and SVF cells will be injected either without modification or after in vitro lipid synthesis induction before transplantation. On average, 12 mice (depending on the amount of tissue) will be transplanted from each tumor specimen donor, and a total of at least 10 patients will be evaluated.

[0148] Experiment 2: Expected Results: The inventors believe that in the absence of scaffolds or adipose-derived cells, the engraftment ("take") rate of tumors alone will be approximately 20%. Furthermore, the time until the tumor physically appears will be prolonged if such tumors are transplanted based on visual detection without the use of scaffolds and / or SVF cells. The Alu-based human genome DNA detection system has proven to be highly sensitive and can detect circulating human tumor cells at least 1-2 months before the physical and visual detection of tumor growth. The presence of Matrigel scaffolds alone is thought to accelerate the rate of (a) tumor growth and (b) engraftment. Mammalian platelet lysates alone are thought to have an effect equivalent to or greater than Matrigel in accelerating tumor growth rate and increasing engraftment rate. The addition of SVF cells further accelerates tumor growth and engraftment rate, to a greater extent than when scaffolds alone are added. Furthermore, the stromal network in the presence of SVF cells is thought to be more vascular and complex compared to the case of scaffolds alone or tumors alone. Finally, the lipid-producing differentiation of scaffolds + SVF cells ("fat on the chip") is thought to promote and enhance tumor growth more than the lipid-producing differentiation of the matrix and / or SVF cells alone, or combinations without lipid-producing differentiation ex vivo.

Claims

1. A biological scaffold comprising mammalian platelet lysate and adipose tissue-derived cell fraction (ATDCF) cells.

2. 2. The biological scaffold of claim 1, wherein the ATDCF cells comprise stromal vascular fraction cells, adipose-derived stromal cells, adipose-derived stem cells, bone marrow-derived mesenchymal stromal cells, and bone marrow-derived mesenchymal stem cells.

3. 3. The biological scaffold of claim 2, wherein the SVF cells are derived from human adipose tissue.

4. 2. The biological scaffold of claim 1, wherein the mammalian platelet lysate is derived from human platelets.

5. 5. The biological scaffold of claim 1, wherein the biological scaffold comprises SVF cells.

6. The biological scaffold according to claim 1 , wherein the biological scaffold comprises at least one of adipose-derived stromal cells and adipose-derived stem cells.

7. 5. The biological scaffold according to claim 1, wherein the biological scaffold comprises at least one of bone marrow-derived mesenchymal stromal cells and bone marrow-derived mesenchymal stem cells.

8. 1. A therapeutic agent comprising a biological scaffold and a substrate, the substrate having at least one surface for application of the biological agent.

9. 10. The method of claim 8, wherein the substrate comprises a tissue culture plate, an orthopedic device, a dental implant, a demineralized bone scaffold, a bandage, a synthetic mesh, a metallic implant, or a ceramic implant.

10. 10. The therapeutic agent of claim 9, wherein the biological scaffold is contacted with the substrate by mixing, seeding, perfusion, filling by capillary action, filling by centrifugation, pouring, printing, or bioprinting of the biological scaffold.

11. 9. The biological scaffold of claim 1, or the therapeutic agent of claim 8, further comprising preadipocytes, vascular smooth muscle cells, endothelial cells and precursors, B and T lymphocytes and precursors, hematopoietic precursors, macrophages, monocyte mesenchymal cells, neurons, neuronal progenitor cells, endothelial cells, endothelial progenitor cells, fibroblasts, dermis, epidermis, cancer-derived cells, cancer stem cells, chondrocytes, muscle cells, adipocytes, osteoblasts, osteoclasts, bone marrow cells, blood cells, and combinations thereof.

12. The therapeutic agent according to claim 8, wherein the substrate is bone.

13. The therapeutic agent of claim 8 , wherein the substrate comprises collagen, adipose extracellular matrix protein, or hPL gel.

14. The therapeutic agent of claim 8 , wherein the substrate is a gel, semi-solid, or solid substrate.

15. 12. The biological scaffold or therapeutic agent of claim 11, wherein the cell is a mammalian cell.

16. 12. The biological scaffold or therapeutic agent of claim 11, wherein the cells are human cells.

17. 12. The biological scaffold or therapeutic agent of claim 11, wherein the cell is a mouse cell.

18. The biological scaffold or therapeutic agent of claim 11 , wherein the cells are adipose-derived stem cells.

19. The biological scaffold or therapeutic agent of claim 11 , wherein the SVF cells are composed of endothelial progenitors.

20. 12. The biological scaffold or therapeutic agent of claim 11, wherein the SVF cells are composed of hematopoietic progenitors.

21. The biological scaffold or therapeutic agent of claim 11 , wherein the SVF cells consist of immune cells.

22. The biological scaffold or therapeutic agent according to claim 11, wherein the SVF cells are vascular smooth muscle cells.

23. The biological scaffold or therapeutic agent according to claim 11 , wherein the SVF cells consist of fibroblasts.

24. 1. A method of producing a biological scaffold-containing formulation or device, the method comprising: Adipose tissue was collected, Isolating ATDCF cells from the adipose tissue; Preparing mammalian platelet lysates combining the ATDCF cells with the mammalian platelet lysate to form a biological scaffold; and applying or contacting the scaffold to at least one surface of a substrate; The method comprises the steps of:

25. The method of claim 24, wherein the ATDCF cells are SVF cells.

26. 25. The method of claim 24, wherein the SVF cells consist of endothelial progenitors.

27. 25. The method of claim 24, wherein the SVF cells consist of hematopoietic progenitors.

28. 25. The method of claim 24, wherein the SVF cells consist of immune cells.

29. 25. The method of claim 24, wherein the SVF cells consist of vascular smooth muscle cells.

30. 29. The method of claim 28, wherein the SVF cells consist of fibroblasts.

31. 26. The method of claim 25, wherein the SVF cells are selected from one or more of preadipocytes, vascular smooth muscle cells, endothelial cells and progenitor cells, B and T lymphocytes and progenitor cells, hematopoietic progenitor cells, macrophages, and monocytes.

32. The method of claim 24, wherein the ATDCF cells comprise one or more of adipose-derived stromal cells and adipose-derived stem cells.

33. The method of claim 24, wherein the ATDCF cells comprise at least one of bone marrow-derived mesenchymal stromal cells and bone marrow-derived mesenchymal stem cells.

34. 25. The method of claim 24, wherein the biological scaffold is applied to or contacted with the at least one surface of the substrate by mixing, seeding, perfusion, capillary action filling, centrifugation filling, pouring, printing, or bioprinting.

35. 29. The method of claim 28, wherein the mammalian platelets are lysed using a freeze / thaw process.

36. 25. The method of claim 24, wherein the mammalian platelets are human platelets.

37. 1. A method of regenerating or repairing tissue in a patient, comprising the steps of: Preparing a biological scaffold; and implanting the biological scaffold into a patient; The method comprising:

38. 1. A method of regenerating or repairing tissue in a patient, comprising the steps of: Preparing a biological scaffold; Incubating the biological scaffold overnight at about 37°C, and implanting the biological scaffold into a patient; The method comprising:

39. 39. The method of claim 37 or 38, wherein the tissue comprises fat, bone, muscle, bone marrow, blood, skin, or cartilage.

40. 39. The method of claim 37 or 38, wherein the tissue is selected from one or more of white adipose tissue, beige adipose tissue, or brown adipose tissue.

41. 39. The method of claim 37 or 38, wherein the patient is a human.

42. 42. The method of any one of claims 37-41, wherein implanting the biological scaffold into the patient further comprises contacting or applying the biological scaffold to at least one surface of a substrate and implanting the substrate into the patient at a site of tissue regeneration or tissue repair.

43. 1. A method for testing the efficacy or toxicity of a drug, said method comprising: Preparing a biological scaffold; contacting the biological scaffold with a drug; Incubating the biological scaffold with the drug; and testing the survival of cells on the biological scaffold; The method comprising:

44. 44. The method of claim 43, wherein the agent comprises a growth factor, a differentiation factor, a cytokine, a chemokine, an adipokine, an antibiotic, an antimycotic, an antifungal agent, an anti-inflammatory agent, or an anti-cancer agent.

45. 1. An immunocompromised or immunodeficient mouse comprising a biological scaffold and a functional human fat pad comprising a recapitulating lesion of a human disease, wherein said mouse is a xenograft mouse.

46. 1. A microfluidic device containing one or more biological scaffolds, wherein the biological scaffolds are isolated and can be subjected to a uniform flow of medium and / or drug.

47. 1. A method for testing the efficacy or toxicity of a drug, said method comprising: Preparing at least two biological scaffolds that are separated from each other and contained in a microfluidic device; treating the microfluidic device with an agent; Incubating the microfluidic device with the agent; and testing the cells for survival in the microfluidic device; The method comprising:

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