Methods and systems for enhancing cells for therapeutic use

Abstract

Methods and systems for enhancing cell populations such as chondrocytes for tissue engineering applications, e.g., the generation of neocartilage. The methods and systems of the present invention feature the introduction of a hypotonic buffer to cells during a cell isolation method, resulting in significantly more mechanically robust neo-tissue (e.g., neo-cartilage) constructs compared to those not treated with a hypotonic buffer. The methods and systems may further include the introduction of cytochalasin D to the hypotonic buffer-purified cells, which can further enhance the mechanical properties and matrix deposition of the cells. The methods and systems result in engineered neo-cartilage from chondrocytes, e.g., fetal tissue, with compressibility comparable to native adult articular cartilage.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Patent Application No. 17 / 496,391, filed October 7, 2021, which is incorporated herein by reference in its entirety.

[0002] government support This invention was made with Government support under Grant No. R01 AR067821 awarded by the NIH. The Government has certain rights in this invention.

[0003] The present invention relates to cell purification methods for applications such as cell and tissue engineering, and cell and tissue transplantation. [Background technology]

[0004] The goal of tissue engineering is to replace damaged tissues to halt disease progression and promote recovery. Primary fully differentiated cells are widely considered to be the ideal cell type for tissue engineering. The cells are phenotypically stable and readily produce tissue-specific extracellular matrix (ECM) molecules. Child and even fetal tissue sources are most desirable due to their enhanced proliferative and synthetic potential compared to adult cells. Tissue engineering products composed of juvenile cells are currently in clinical use. For example, RevaFlex (ISTO Technologies), a tissue engineering product for cartilage repair using juvenile chondrocytes, is currently in Phase III clinical trials in the United States. Although these engineered tissues show promise, they do not yet recapitulate the properties and structure of native tissues.

[0005] Primary fully differentiated cells from children and fetuses are widely considered to be the ideal cell type for tissue engineering applications. However, their use in tissue engineering can be hindered by contamination with undesirable cell types that prevent the cells from achieving similar functional properties as those produced from adult levels of cells or healthy cells. Increasing the mechanical properties of neocartilage from fetal chondrocytes to adult levels has not been achieved so far.

[0006] Tissue engineering efforts using primary cells can be hampered by contamination with undesirable cell types. Contamination with blood and surrounding tissues can occur during isolation of the target donor tissue. Furthermore, many tissues are composed of multiple cell types, not all of which are suitable for tissue engineering applications. In addition, disease states and tissue maturation can introduce undesirable cell phenotypes into the isolated population. Aging tissues, which are prone to diseases such as cancer, atherosclerosis, and osteoarthritis, contain senescent cells that increasingly produce reactive oxygen species, inflammatory mediators, and matrix-degrading enzymes. These limitations necessitate the use of cell purification methods during isolation to eliminate the presence of undesirable phenotypes (e.g., undesirable cytoskeletal properties) to achieve a homogenous cell population enriched for cells with properties suitable for tissue engineering. Summary of the Invention

[0007] The present invention features methods and systems for improving cells for therapeutic use, such as cell purification methods that enhance cell populations by enriching for cells with properties conducive to cell and tissue engineering.

[0008] Articular cartilage tissue engineering is well established and can be used as an exemplary system. However, although typically unrecognized, undesirable cell phenotypes in chondrocytes may be present due to several reasons. Contamination by hematopoietic cells (e.g., pro-apoptotic cells), or cells from other surrounding tissues, may occur when performing cartilage biopsies for clinical applications such as autologous chondrocyte implantation (ACI). Short-term exposure of cartilage to blood has been shown to induce chondrocyte apoptosis in models reflecting hemophilia. Second, in clinical settings, autologous or allogeneic cartilage grafts are often taken from adult tissues that exhibit matrix degradation, surface defects, and fibrillation. Diseased cartilage, such as osteoarthritis, experiences enhanced ECM degeneration and contains chondrocytes of altered phenotype. Degenerative changes to the cartilage ECM are associated with chondrocyte apoptosis. Fetal cartilage, on the other hand, is vascularized, thus introducing blood and a plethora of cell types into the mass of tissue from which chondrocytes are isolated. In addition, even in healthy tissue, the isolation of cartilage itself induces tissue damage, resulting in necrosis at the wound edges and a wave of apoptosis spreading throughout the tissue. In addition to red blood cell (RBC) contamination, cell phenotypic heterogeneity due to altered phenotype of chondrocytes is an unexpected factor limiting the ability of engineered cartilage properties to reach those of native tissue.

[0009] Despite the possibility of contamination during chondrocyte isolation, only a few studies have attempted to demonstrate its importance. Using collagenase to sequentially digest whole hamster rib cartilage into two fractions, it was demonstrated that the second fraction contained a cell population with a more homogenous chondrocyte morphology compared to the whole unseparated population. Another method of purifying isolated chondrocytes is by sequential plating. Rat chondrocyte isolates separated by differential adherence to tissue culture plastic showed 100% chondrocytes after the eighth plating versus a mixture of cells when the whole population was plated. Yet another method suggests the use of cell surface markers such as CD14 and CD45 to exclude contamination by monocytes and hematopoietic cells. Ammonium chloride-potassium lysis buffer (ACK buffer) is commonly used to lyse RBCs in samples containing white blood cells such as EDTA-treated whole blood, buffy coat, and bone marrow. For tissue engineering purposes, ACK buffer has been used to isolate pure populations of stem cells, such as adipose-derived stem cells and mesenchymal stem cells, but has not yet been explored in the isolation of non-stem cell types (e.g., cartilage). Because contaminating cell types in many isolations of fully differentiated cells may include cells with alternative phenotypes, ACK buffer treatment holds promise for the purification of cell populations desirable for tissue engineering applications. Although ACK buffer treatment may lyse all cells, the present invention allows for the preferential destruction of cells with altered phenotypes (e.g., pre-apoptotic cells) to enrich for cells with the preferred phenotype for new tissue formation.

[0010] One of the unique and inventive technical features of the present invention is the use of hypotonic solutions (e.g., ACK buffer) to treat freshly isolated, fully differentiated cells to remove pre-apoptotic cells and enhance their ability to form biofunctional tissue. Without wishing to limit the present invention to any theory or mechanism, it is believed that the methods and systems of the present invention can improve the mechanical properties of new tissues made from specific cell populations (e.g., fetal age cells, diseased tissue sources) to those made from adult level cells or healthy cells.

[0011] Furthermore, the prior art teaches away from the present invention. For example, the prior art utilizes hypotonic solution treatment on cells sourced from fetal sheep or young cows, but does not show the use of hypotonic solution treatment on cells sourced from humans to remove pre-apoptotic cells. Figure 18 shows that a treatment that is beneficial for cells from one species (e.g. fetal sheep) is not necessarily beneficial when applied to cells from another species (e.g. human). Therefore, it is not self-evident that the same treatment (e.g. hypotonic solution) will work similarly on cells from different species.

[0012] In addition, Figure 19 shows that even within the same species (e.g., juvenile Yucatan minipigs), different cell types responded differently to the same treatment or culture regimen. Thus, it is not self-evident that a treatment or culture regimen beneficial to one cell type (e.g., articular chondrocytes) can be directly applied to another cell type (e.g., rib chondrocytes) to achieve the same beneficial effect.

[0013] Surprising results Since the prior art teaches the use of hypotonic buffer treatment on cell populations that contain blood cells, it is surprising that cells isolated from non-vascular tissues, such as cartilage, are responsive to ACK buffer treatment.

[0014] Furthermore, while the prior art has indicated the use of ACK buffer treatment on stem cells, chondrocytes, which do not contain blood cells, respond to ACK buffer treatment in an unexpected manner by forming engineered neocartilage.

[0015] It was surprising that by subjecting chondrocytes to a hypotonic buffer such as ACK buffer, which selects for cells with pre-existing undesirable cytoskeletal properties, undesirable membrane properties, and altered stiffness, an enriched population of cells was obtained.

[0016] It was surprising that cells with undesirable, e.g. pro-apoptotic properties, are present in young healthy cartilage to such an extent that the formation of engineered neocartilage is affected by their presence.

[0017] Surprisingly, it has been discovered that the methods and systems of the present invention result in engineered scaffold-free neocartilage from enriched fully differentiated cells resulting from the processes described herein, which achieves compressibility comparable to native adult articular cartilage. Increasing the mechanical properties of neocartilage from fetal chondrocytes to adult levels has not been achieved before. The present invention features a method to enrich cell populations suitable for neocartilage development, and further enables methods to engineer cytoskeleton to enhance cells for therapeutic use. For example, the use of hypotonic buffers during chondrocyte purification significantly improved the uniformity, matrix deposition, and mechanical properties of neocartilage constructs. The combination of hypotonic buffers and cytochalasin D results in engineered neocartilage from fetal age chondrocytes, which achieves compressibility comparable to native adult articular cartilage. Without wishing to limit the present invention to any particular theory or mechanism, it is believed that by reducing RBC contamination, as well as removing chondrocytes of altered phenotype that are cellular detractors to the self-assembly process, and eliminating apoptotic stimuli, uniformity of the neo-cartilage, chondrocyte distribution, and ECM deposition within the neo-cartilage are improved, thereby enhancing the biochemical and mechanical properties of the engineered tissue formed by the treated cells.

[0018] These results are surprising as this level of mechanical robustness has not previously been seen with a fetal chondrocyte source.

[0019] The invention features methods for preparing cells or cell populations and enriching cell populations for therapeutic use. The invention also features methods for preparing tissues and enriching tissues for therapeutic use.

[0020] The present invention features a method of enriching a sample of chondrocytes (e.g., a sample of human chondrocytes). The method can include obtaining a sample of chondrocytes from cartilage tissue; and subjecting the sample of chondrocytes to treatment with a hypotonic solution (e.g., ammonium chloride potassium lysis (ACK) buffer). In some embodiments, the method can be repeated multiple times, alone or in combination with other treatments.

[0021] In some embodiments, the invention features a method of preparing a sample of chondrocytes (e.g., human chondrocytes). The method can include obtaining a sample of chondrocytes (e.g., human chondrocytes), where the sample of chondrocytes (e.g., human chondrocytes) includes a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and subjecting the sample of chondrocytes to a treatment (e.g., the treatment includes a hypotonic solution (e.g., ACK)). In some embodiments, the method can be repeated multiple times, alone or in combination with other treatments.

[0022] The invention further features a method of preparing a sample of human chondrocytes, which may include obtaining a sample of human chondrocytes sourced from a portion of a rib and subjecting the sample of human chondrocytes to treatment with a hypotonic solution, such as ammonium chloride potassium lysis (ACK) buffer.

[0023] The invention may also feature a treated cell sample produced from a method that includes (a) obtaining a sample of cartilage and digesting the cartilage sample to obtain a cell suspension, and (b) subjecting the cell suspension to a hypotonic solution to obtain a treated cell sample. In some embodiments, the treated cell sample is suitable for neocartilage generation.

[0024] The present invention features a method of enriching a cell population, comprising: 1) obtaining a population of somatic cells; 2) subjecting the population of somatic cells to a treatment that selects for cells with pre-existing undesirable characteristics (e.g., pre-existing undesirable cytoskeleton characteristics, pre-existing undesirable membrane surface area characteristics, or pre-existing altered stiffness characteristics); 3) isolating and removing cells with pre-existing undesirable characteristics; and 4) isolating and retaining the remaining cell population enriched for cells without pre-existing undesirable characteristics (e.g., pre-existing undesirable cytoskeleton characteristics, pre-existing undesirable membrane surface area characteristics, or pre-existing altered stiffness characteristics). These steps can be repeated multiple times, alone or in combination with other treatments.

[0025] Any feature or combination of features described herein is encompassed within the scope of the present invention, unless the features encompassed in any such combination are mutually inconsistent, as becomes apparent from the context, the specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.

[0026] The features and advantages of the present invention will become apparent from consideration of the following detailed description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0027] [Figure 1] Figure 1 shows pellet morphology, viability, and red blood cell (RBC) content of fetal ovine AC and young bovine AC before and after ACK treatment. ACK treatment resulted in a change in cell pellet color and a significant decrease in RBC content.

[0028] [Figure 2A] Overall neocartilage morphology and selected parameters are shown in Figure 2A, which shows that ACK treatment eliminated the bulbous diffuse region (indicated by white arrow) in fetal ovine AC neocartilage. [Figure 2B] Overall neocartilage morphology and selected parameters are shown in Figure 2B. Figure 2B shows that ACK treatment reduced the thickness of fetal ovine neocartilage. [Figure 2C] Overall neocartilage morphology and selected parameters are shown in Figure 2C, which shows that ACK treatment reduced the wet weight of fetal ovine neocartilage. [Figure 2D] Neocartilage gross morphology and select parameters are shown. Figure 2D shows that ACK treatment did not affect hydration in fetal sheep. [Figure 2E] Overall neocartilage morphology and selected parameters are shown in Figure 2E, which shows that ACK treatment eliminated the bulbous diffuse regions (indicated by white arrows) in juvenile bovine AC neocartilage. [Figure 2F] The overall neocartilage morphology and selected parameters are shown in Figure 2F, which shows that ACK treatment reduced the thickness of young bovine neocartilage. [Figure 2G] The overall neocartilage morphology and selected parameters are shown in Figure 2G, which shows that ACK treatment reduced the wet weight of young bovine neocartilage. [Figure 2H] Neocartilage whole body morphology and select parameters are shown. Figure 2H shows that ACK treatment did not affect hydration in young bovine cartilage.

[0029] [Diagram 3] Neocartilage histology is shown. ACK treatment of fetal ovine and young bovine AC eliminated the hypocellular, diffuse areas present in untreated constructs (*), enhanced the homogeneity of the neocartilage, and enhanced GAG, total collagen, and collagen II staining.

[0030] [Figure 4A] The biochemical content of neocartilage in fetal ovine AC (foAC) and young bovine AC (jbAC) with and without ACK treatment is shown in Figure 4. Figure 4A shows that ACK treatment significantly reduced caspase activity in foAC. [Figure 4B] Figure 4B shows the biochemical content of neocartilage in fetal ovine AC (foAC) and young bovine AC (jbAC) with and without ACK treatment. Figure 4B shows that ACK treatment did not affect the GAG / WW content in foAC. [Figure 4C] Figure 4 shows the biochemical content of neocartilage in fetal ovine AC (foAC) and young bovine AC (jbAC) with and without ACK treatment. Figure 4C shows that ACK treatment did not affect the GAG / DW content in foAC. [Figure 4D] Figure 4 shows the biochemical content of neocartilage in fetal ovine AC (foAC) and young bovine AC (jbAC) with and without ACK treatment. Figure 4D shows that ACK treatment significantly increased collagen / WW content in foAC. [Figure 4E] Figure 4 shows the biochemical content of neocartilage in fetal ovine AC (foAC) and young bovine AC (jbAC) with and without ACK treatment. Figure 4E shows that ACK treatment significantly increased collagen / DW content in foAC. [Figure 4F] The biochemical content of neocartilage in fetal ovine AC (foAC) and young bovine AC (jbAC) with and without ACK treatment is shown in Figure 4F, which shows that ACK treatment significantly reduced caspase activity in jbAC. [Figure 4G] Figure 4G shows the biochemical content of neocartilage in fetal ovine AC (foAC) and young bovine AC (jbAC) with and without ACK treatment. ACK treatment significantly reduced the GAG / WW content in jbAC. [Figure 4H] Figure 4 shows the biochemical content of neocartilage in fetal ovine AC (foAC) and young bovine AC (jbAC) with and without ACK treatment. Figure 4H shows that ACK treatment significantly reduced GAG / DW content in jbAC. [Figure 4I] Figure 4 shows the biochemical content of neocartilage in fetal ovine AC (foAC) and young bovine AC (jbAC) with and without ACK treatment. Figure 4I shows that ACK treatment significantly increased collagen / WW content in jbAC. [Figure 4J]Figure 4 shows the biochemical content of neocartilage in fetal ovine AC (foAC) and young bovine AC (jbAC) with and without ACK treatment. Figure 4J shows that ACK treatment did not affect the GAG / WW content in jbAC.

[0031] [Diagram 5] Mechanical properties of neocartilage. ACK treatment significantly increased all mechanical properties measured for both cell types.

[0032] [Figure 6A] The effect of seeding density on the gross morphology, biochemical content, and histology of neocartilage is shown in Figure 6. Figure 6A shows that gross abnormalities appear at seeding densities of 5 and 4 million cells at passages P0 and P3R, respectively. [Figure 6B] The effect of seeding density on the gross morphology, biochemical content, and histology of neocartilage is shown. Figure 6B shows that the GAG / DN of P3R neocartilage exhibits a seeding density dependent effect and exceeds that of P0 neocartilage. [Figure 6C] The effect of seeding density on the gross morphology, biochemical content, and histology of neocartilage is shown. Figure 6C shows that the mechanical property aggregate modulus increases with seeding density of P0 cells and decreases with seeding density of P3R cells. [Figure 6D] The effect of seeding density on the gross morphology, biochemical content, and histology of neocartilage is shown. Figure 6D shows that the collagen / DNA of P3R neocartilage exhibits a seeding density dependent effect and exceeds that of P0 neocartilage. [Figure 6E] The effect of seeding density on the gross morphology, biochemical content, and histology of neocartilage is shown in Figure 6E, which shows that the mechanical property tensile modulus increases with the seeding density of P0 cells and decreases with the seeding density of P3R cells. [Figure 6F] The effect of seeding density on the gross morphology, biochemical content, and histology of neocartilage is shown. Figure 6F shows that the pyridinoline content of P0 neocartilage exceeds that of P3R neocartilage. [Figure 6G]The effect of seeding density on the gross morphology, biochemical content, and histology of neocartilage is shown. Figure 6G shows that the mechanical property ultimate tensile strength increases with seeding density of P0 cells and decreases with seeding density of P3R cells. [Figure 6H] The effect of seeding density on the gross morphology, biochemical content, and histology of neocartilage is shown. Figure 6H shows H&E and immunohistochemistry (IHC) staining for GAGs, type I collagen (col I), type II collagen (col II), and total collagen (total col). IHC controls are meniscus (M), articular cartilage (AC), and tendon (T). (Phase 1).

[0033] [Figure 7] Phenotypic confirmation of engineered neocartilage is shown. Histological controls are articular cartilage (AC) and growth plate (GP).

[0034] [Figure 8A] Figure 8 shows the effect of cytochalasin D (Cyto D) and hyaluronidase (Hya) treatment of P3R neocartilage. Figure 8A shows that gross abnormalities were only present in the Hya-treated group. [Figure 8B] FIG. 8B shows the effect of cytochalasin D (Cyto D) and hyaluronidase (Hya) treatment of P3R neocartilage. FIG. 8B shows H&E and IHC staining for GAGs, type I collagen (col I), type II collagen (col II), and total collagen (total col). IHC controls are meniscus (M), articular cartilage (AC), and tendon (T). (Phase 2). [Figure 8C] Figure 8C shows the effect of cytochalasin D (Cyto D) and hyaluronidase (Hya) treatment of P3R neocartilage. Figure 8C shows that GAG / wet weight and mechanical properties were increased by Cyto D treatment. [Figure 8D] Figure 8 shows the effect of cytochalasin D (Cyto D) and hyaluronidase (Hya) treatment of P3R neocartilage. Figure 8D shows that aggregate modulus was increased by Cyto D treatment. [Figure 8E]Figure 8E shows the effect of cytochalasin D (Cyto D) and hyaluronidase (Hya) treatment of P3R neocartilage. Figure 8E shows that collagen content was unchanged by either treatment. [Figure 8F] Figure 8F shows the effect of cytochalasin D (Cyto D) and hyaluronidase (Hya) treatment of P3R neocartilage. Tensile modulus was increased by Cyto D treatment. [Figure 8G] Figure 8G shows the effect of cytochalasin D (Cyto D) and hyaluronidase (Hya) treatment of P3R neocartilage, and shows that pyridinoline content was unchanged by either treatment. [Figure 8H] Figure 8H shows the effect of cytochalasin D (Cyto D) and hyaluronidase (Hya) treatment of P3R neocartilage. Ultimate tensile strength was increased by Cyto D treatment.

[0035] [Figure 9] Figure 1 shows the effect of cytochalasin D treatment on actin arrangement. Cytochalasin D treatment resulted in enhanced cortical arrangement of actin in both P3 and P3R chondrocytes (phase 2).

[0036] [Figure 10A] Figure 10 shows the effect of Cytochalasin D (Cyto D) and TCL treatment of P3R neocartilage. Figure 10A shows no gross abnormalities. [Figure 10B] The effect of cytochalasin D (Cyto D) and TCL treatment of P3R neocartilage is shown. Figure 10B shows H&E and IHC staining for GAG, type I collagen (col I), type II collagen (col II), and total collagen (total col). IHC controls are meniscus (M), articular cartilage (AC), and tendon (T). [Figure 10C] The effect of Cytochalasin D (Cyto D) and TCL treatment of P3R neocartilage is shown in Figure 10C, which shows that no significant differences were observed in GAG content. [Figure 10D]Figure 10D shows the effect of Cytochalasin D (Cyto D) and TCL treatment of P3R neocartilage, showing that no significant differences in aggregation were observed. [Figure 10E] Figure 10 shows the effect of cytochalasin D (Cyto D) and TCL treatment of P3R neocartilage. Figure 10E shows that TCL treatment in combination with Cyto D (Cyto D+TCL) increased collagen content. [Figure 10F] Figure 10 shows the effect of cytochalasin D (Cyto D) and TCL treatment of P3R neocartilage. Figure 10F shows that TCL treatment in combination with Cyto D (Cyto D+TCL) increased tensile stiffness (phase 3). [Figure 10G] Figure 10 shows the effect of cytochalasin D (Cyto D) and TCL treatment of P3R neocartilage. Figure 10G shows that TCL treatment in combination with Cyto D (Cyto D+TCL) increased pyridinoline content. [Figure 10H] Figure 10 shows the effect of cytochalasin D (Cyto D) and TCL treatment of P3R neocartilage. Figure 10H shows that TCL treatment in combination with Cyto D (Cyto D+TCL) increased strength (phase 3).

[0037] [Figure 11A] The results show an increase in neocartilage functional properties. Figure 11A shows that aggregate modulus increased 9.6-fold. [Figure 11B] This shows an increase in neocartilage functional properties. Figure 11B shows that the shear modulus was increased by 7.2 fold. [Figure 11C] This shows an increase in neocartilage functional properties. Figure 11C shows that the tensile modulus increased 3.8-fold. [Figure 11D] This shows an increase in neocartilage functional properties: Figure 11D shows that the ultimate tensile strength was increased by 9.0 fold. [Figure 11E] Figure 11E shows that P3R neocartilage exceeded fetal and juvenile native tissue values ​​and approached adult levels (phases 1-3).

[0038] [Figure 12A] Figure 12 shows the effect of cytochalasin D (Cyto D) and hyaluronidase (Hya) treatment of P3 neocartilage. Figure 12A shows that Cyto D treatment resulted in only flat constructs. [Figure 12B] The effect of cytochalasin D (Cyto D) and hyaluronidase (Hya) treatment of P3 neocartilage is shown. Figure 12B shows H&E and IHC staining for GAGs, type I collagen (col I), type II collagen (col II), and total collagen (total col). IHC controls (B) are meniscus (M), articular cartilage (AC), and tendon (T) (phase 2).

[0039] [Figure 13] Table 1 (data from Phase 1) is shown. Data are presented as mean ± standard deviation. Statistics were calculated across groups within biochemical or mechanical parameters. Statistical significance is indicated within groups marked with different letters.

[0040] [Figure 14] Table 2 (data from Phase 2, P3) is shown. Data are presented as mean ± standard deviation. Statistics were calculated across groups within biochemical or mechanical parameters. Statistical significance is indicated within groups, marked with different letters.

[0041] [Figure 15] Table 3 (Phase 2) is shown. Data are presented as mean ± standard deviation. Statistics were calculated across groups within biochemical or mechanical parameters. Statistical significance is indicated within groups marked with different letters.

[0042] [Figure 16] Table 4 (Phase 3) is shown. Data are presented as mean ± standard deviation. Statistics were calculated across groups within biochemical or mechanical parameters. Statistical significance is indicated within groups marked with different letters.

[0043] [Figure 17]A summary of compressibility is shown: Aggregate modulus of ACK buffer-treated P3R cells seeded at optimal density with cytochalasin D was increased 9.6-fold over P0 controls.

[0044] [Figure 18] Chondrocytes sourced from either fetal ovine articular cartilage or adult human costal cartilage were passaged three times (P3) to undergo aggregate redifferentiation (rejuvenation) and seeded at 2 million cells per neo-cartilage construct using the self-assembly method. Control constructs were untreated. Constructs treated with cytochalasin D (Cyto D) were treated with 2 μM on days 0-2. When Cyto D was used on neo-cartilage constructs formed with fetal ovine articular chondrocytes (foAC), both the compressive aggregate modulus and the shear modulus were significantly increased. This is beneficial. However, in stark contrast, when Cyto D was used on adult human costal chondrocytes (ahCC), the aggregate modulus and shear modulus were significantly decreased. This example clearly shows that a treatment beneficial for cells from one species is not self-evident that it will be beneficial when used on cells from another species.

[0045] [Figure 19]Chondrocytes sourced from either juvenile Yucatan minipig articular cartilage (jyAC) or juvenile Yucatan minipig costal cartilage (jyCC) were passaged three times (P3) to undergo aggregate redifferentiation (rejuvenation). For the control, 2 million jyAC or jyCC were allowed to self-assemble to form neocartilage constructs. For the experimental groups, 2 million jyAC or jyCC were first subjected to neocartilage self-assembly and then seeded on top with a second 2 million cells 1, 2, 3, or 4 hours later in order to increase the thickness of the construct. jyAC-derived neocartilage with a second layer of cells added at 1, 2, 3, and 4 hours was significantly thicker than the control construct and the diameter of the construct was unaffected. However, for jyCC-derived neocartilage, the thickness was not affected by the additional layer of cells added at any time point. Furthermore, the diameter of the construct decreased significantly with the addition of cells per time point. Thus, it is not obvious that a treatment or culture regimen beneficial to one cell type can be directly used on another cell type to achieve a beneficial effect, even within the same species.

[0046] [Figure 20] ACK-treated human rib chondrocytes from adult and juvenile donors are shown. Chondrocytes sourced from either adult or pediatric rib cartilage were treated with a hypotonic solution (ACK buffer) according to the methods described herein. Briefly, the entire population of cells freshly isolated from rib tissue was subjected to ACK treatment for 10 minutes to enrich the population of non-pre-apoptotic cells. This fraction of non-pre-apoptotic cells was then allowed to undergo proliferation, aggregate redifferentiation (rejuvenation), and self-assembly up to passage 3 (P3) at 2 million cells per neo-cartilage construct. Results: ACK-treated human rib chondrocytes sourced from adult and juvenile donors each generated morphologically accurate (e.g., flat and not curled) neo-cartilage constructs. The neo-cartilage constructs were uniform in thickness and contained phenotypically accurate extracellular matrix and viable chondrocytes. The constructs also exhibited robust compressive (aggregate modulus) and tensile (tensile modulus) mechanical properties.

[0047] term Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs.

[0048] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. Furthermore, wherever the terms "including," "includes," "having," "has," "with," or variations thereof, are used in either the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0049] As used herein, "apoptosis" refers to a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cellular changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, DNA fragmentation, and mRNA degradation.

[0050] As used herein, "pre-apoptosis" refers to an altered state that is distinct from apoptosis. In some embodiments, pre-apoptotic cells share some characteristics with apoptotic cells. However, pre-apoptosis is reversible, and apoptosis must be induced in addition to the method for the cell to die. Characteristics of pre-apoptotic cells may include, but are not limited to, a reduction in cell membrane surface area, changes in cell stiffness (e.g., undesirable stiffness properties, see below), or changes in the cytoskeleton (e.g., undesirable cytoskeletal properties, see below). In some embodiments, pre-apoptosis and apoptosis are two different cellular states. In other embodiments, pre-apoptotic and apoptotic are two different cellular states.

[0051] As used herein, "pro-apoptotic" refers to proteins and / or cells that can cause other cells to become pre-apoptotic or apoptotic.

[0052] "Enrichment," as used herein, refers to the generation of cell fractions (e.g., samples of cells after processing) with improved cellular homogeneity, with properties suitable for cell / tissue engineering, improved cellular robustness, improved cell phenotype, and improved properties leading to improved tissue engineering.

[0053] As used herein, an "undesirable cell type" may refer to cells that are not suitable for tissue engineering applications, such as pre-apoptotic cells or pro-apoptotic cells (eg, red blood cells).

[0054] As used herein, "undesirable cytoskeletal properties" refers to cells with a weakened, fragmented, disrupted or modified cytoskeleton, cells with a cytoskeleton that is unable to remodel or has reduced remodeling capacity, cells with cytoskeletal properties that make the cells more susceptible to disruption by treatment (e.g., treatment with a hypotonic solution such as ACK buffer), or cells with a combination of the foregoing characteristics.

[0055] As used herein, "undesirable membrane properties" refers to cells with reduced membrane surface area, cells with disrupted or altered membranes, cells whose membranes cannot adapt to conformational changes / size changes, cells with membrane properties that make the cells more susceptible to disruption by treatment (e.g., treatment with hypotonic solutions such as ACK buffer), or cells with a combination of the aforementioned characteristics.

[0056] As used herein, "undesirable stiffness properties" refers to cells that have reduced overall stiffness, cells that have increased overall stiffness, cells whose stiffness varies depending on the region of the cell being examined, cells that have reduced flexibility, cells that have stiffness that makes the cells more susceptible to disruption by treatment (e.g., treatment with a hypotonic solution such as ACK buffer), or cells that have a combination of the aforementioned characteristics.

[0057] As used herein, "undesirable" refers to a cellular characteristic that has a detrimental effect. For example, cells with undesirable characteristics (including, but not limited to, undesirable cytoskeleton characteristics, undesirable membrane characteristics, undesirable stiffness characteristics, or a combination thereof) are less likely to respond appropriately to various stresses and therefore more susceptible to death (e.g., detrimental effects). In addition, cells with undesirable characteristics may produce extracellular matrix with altered composition (e.g., collagen I instead of collagen II), less extracellular matrix overall, or extracellular matrix with altered mechanical properties (e.g., reduced stiffness and strength).

[0058] As used herein, a "pre-existing" characteristic (e.g., property) refers to a characteristic of a cell (or cell population) that is present during or after collection of a sample of cells, but is not present after processing of the cell population (or cells) by the methods described herein.

[0059] As used herein, "chondrocytes" are the only cells found in healthy cartilage that generate and maintain the cartilage matrix, which may include collagen and proteoglycans.

[0060] "Cartilage" as used herein is a non-vascular type of supporting connective tissue found throughout the body. There are three types of cartilage: hyaline (e.g., non-articular cartilage such as rib cartilage), fibrocartilage, and elastic cartilage. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] For purposes of summarizing the disclosure, certain aspects, advantages, and novel features of the disclosure are described herein. It should be understood that not all such advantages may necessarily be achieved in accordance with any particular embodiment of the disclosure. Thus, the disclosure may be implemented or performed to achieve or optimize one advantage or group of advantages as taught herein, without necessarily achieving other advantages as may be taught or suggested herein.

[0062] In addition, although the embodiments of the present disclosure have been described in detail, certain variations and modifications will be apparent to those skilled in the art, including embodiments that do not realize all of the features and benefits described herein. It will be understood by those skilled in the art that the present disclosure extends beyond the specifically disclosed embodiments to other alternative or additional embodiments and / or uses thereof, as well as obvious modifications and equivalents. Furthermore, while numerous variations have been shown and described in various details, other modifications that are within the scope of the present disclosure will be readily apparent to those skilled in the art based on the present disclosure. It is also intended that various combinations or subcombinations of specific features and aspects of the embodiments may be made and still fall within the scope of the present disclosure. Thus, it should be understood that various features and aspects of the disclosed embodiments can be combined with or substituted for each other to form various forms of the present disclosure. It is therefore intended that the scope of the present disclosure disclosed herein should not be limited by the specific disclosed embodiments described herein.

[0063] The invention features a method of preparing (e.g., enriching) a sample of chondrocytes (e.g., a sample of human chondrocytes). The method can include obtaining a sample of chondrocytes from cartilage tissue; and subjecting the sample of chondrocytes to treatment with a hypotonic solution (e.g., ammonium chloride potassium lysis (ACK) buffer). In other embodiments, the method can be repeated multiple times, alone or in combination with other treatments.

[0064] The invention may also feature a method of preparing (e.g., enriching) a sample of non-articular chondrocytes. The method may include obtaining a sample of non-articular chondrocytes from cartilage tissue; and subjecting the sample of non-articular chondrocytes to treatment with a hypotonic solution (e.g., ammonium chloride potassium lysis (ACK) buffer). In other embodiments, the method may be repeated multiple times, alone or in combination with other treatments.

[0065] The invention may feature a method of preparing (e.g., enriching) a sample of chondrocytes (e.g., human chondrocytes). The method may include obtaining a sample of chondrocytes (e.g., human chondrocytes) and subjecting the sample of chondrocytes to a treatment (e.g., a hypotonic solution, e.g., ACK buffer). In other embodiments, the method includes obtaining a sample of chondrocytes (e.g., human chondrocytes) and subjecting the sample of chondrocytes to a treatment with a hypotonic solution (e.g., ACK). In some embodiments, the sample of chondrocytes (e.g., human chondrocytes) comprises a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes. In other embodiments, the method may be repeated multiple times, alone or in combination with other treatments.

[0066] In some embodiments, the treated chondrocyte (e.g., human chondrocyte) sample has a higher percentage of non-pre-apoptotic cells compared to the treated chondrocyte sample. In some embodiments, the treated chondrocyte (e.g., human chondrocyte) sample has a lower percentage of pre-apoptotic cells compared to the treated chondrocyte sample. In some embodiments, the treated chondrocyte (e.g., human chondrocyte) sample has a lower percentage of pro-apoptotic cells compared to the treated chondrocyte sample. In other embodiments, the treated chondrocyte (e.g., human chondrocyte) sample has a lower percentage of undesirable cell types compared to the treated chondrocyte sample.

[0067] The invention may also feature a method of preparing (e.g., enriching) a cell population. The method may include obtaining a sample of cells sourced from a portion of a rib and subjecting the sample of cells sourced from the portion of a rib to a treatment (e.g., a hypotonic solution, e.g., ACK buffer). In other embodiments, the method includes obtaining a sample of cells sourced from a portion of a rib and subjecting the sample of cells sourced from the portion of a rib to a treatment with a hypotonic solution (e.g., ACK). In some embodiments, the sample of cells from the portion of a rib includes a mixed population of non-pre-apoptotic cells and pre-apoptotic cells. In other embodiments, the method may be repeated multiple times, alone or in combination with other treatments.

[0068] In some embodiments, the sample of cells sourced from the processed rib portion has a higher percentage of non-pre-apoptotic cells compared to the sample of cells sourced from the rib portion before processing. In some embodiments, the sample of cells sourced from the processed rib portion has a lower percentage of pre-apoptotic cells compared to the sample of cells sourced from the rib portion before processing. In some embodiments, the sample of cells sourced from the processed rib portion has a lower percentage of pro-apoptotic cells compared to the sample of cells sourced from the rib portion before processing. In other embodiments, the sample of cells sourced from the processed rib portion has a lower percentage of undesirable cell types compared to the sample of cells sourced from the rib portion before processing.

[0069] The invention may further feature a method of preparing (e.g., enriching) a sample of non-articular chondrocytes. The method may include obtaining a sample of non-articular chondrocytes and subjecting the sample of non-articular chondrocytes to a treatment (e.g., a hypotonic solution, e.g., ACK buffer). In other embodiments, the method includes obtaining a sample of non-articular chondrocytes and subjecting the sample of non-articular chondrocytes to a treatment with a hypotonic solution (e.g., ACK). In some embodiments, the sample of non-articular chondrocytes comprises a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes. In some embodiments, the method may be repeated multiple times, alone or in combination with other treatments.

[0070] In some embodiments, the treated sample of non-articular chondrocytes has a higher percentage of non-pre-apoptotic cells compared to the treated sample of non-articular chondrocytes. In some embodiments, the treated sample of non-articular chondrocytes has a lower percentage of pre-apoptotic cells compared to the treated sample of non-articular chondrocytes. In some embodiments, the treated sample of non-articular chondrocytes has a lower percentage of pro-apoptotic cells compared to the treated sample of non-articular chondrocytes. In other embodiments, the treated sample of non-articular chondrocytes has a lower percentage of undesirable cell types compared to the treated sample of non-articular chondrocytes.

[0071] The invention also features a method of preparing (e.g., enriching) a human cell population. The method can include obtaining a sample of human cells sourced from a portion of a rib and subjecting the sample of human cells to a treatment (e.g., a hypotonic solution, e.g., ACK buffer). In other embodiments, the method includes obtaining a sample of human cells sourced from a portion of a rib and subjecting the sample of cells to a treatment with a hypotonic solution (e.g., ACK). In other embodiments, the method can be repeated multiple times, alone or in combination with other treatments. In some embodiments, the sample of cells from the portion of a rib includes a mixed population of non-pre-apoptotic cells and pre-apoptotic cells. In other embodiments, the method can be repeated multiple times, alone or in combination with other treatments.

[0072] In some embodiments, the methods described herein further comprise passaging the cells in a monolayer or three-dimensional environment after treatment, hi other embodiments, the methods described herein further comprise generating neocartilage with the passaged cells.

[0073] In some embodiments, the sample comprises chondrocytes. In other embodiments, the sample comprises human chondrocytes. In some embodiments, the sample comprises non-articular chondrocytes. In other embodiments, the sample comprises human non-articular chondrocytes. In some embodiments, the sample comprises cells derived from cartilage, for example, the sample may be derived from any cartilage, including but not limited to hyaline cartilage, fibrocartilage, elastic cartilage. In some embodiments, the sample comprises cells derived from a portion of a rib. In other embodiments, the sample comprises cells derived from a portion of a human rib. In some embodiments, the portion of the rib comprises rib chondrocytes. In other embodiments, the portion of the rib comprises rib tissue. In further embodiments, the portion of the rib comprises rib chondrocytes. In some embodiments, the rib tissue comprises chondrocytes.

[0074] In some embodiments, the sample of cells is a sample of chondrocytes. In other embodiments, the sample of cells is a sample of non-articular chondrocytes. In some embodiments, the sample of cells is human cells. In other embodiments, the sample of cells is from a portion of a rib. In some embodiments, the rib comprises chondrocytes. In some embodiments, the chondrocytes are non-articular chondrocytes.

[0075] In some embodiments, the treatment comprises adding a hypotonic solution to the cell sample to induce cell swelling. Any hypotonic solution may be used according to the methods described herein, such as potassium ammonium chloride lysis buffer (ACK buffer) or water. In some embodiments, the hypotonic solution is potassium ammonium chloride lysis buffer (ACK buffer). In other embodiments, the treatment comprises adding a hypotonic solution to the obtained cell sample. In some embodiments, the treatment induces cell swelling. In some embodiments, the hypotonic solution induces cell swelling. In some embodiments, the cell swelling causes cell death. In some embodiments, the treatment preferentially induces swelling-associated death of pre-apoptotic cells. In other embodiments, the hypotonic solution preferentially induces swelling-associated death of pre-apoptotic cells. In further embodiments, the ACK buffer preferentially induces swelling of pre-apoptotic cells.

[0076] Without wishing to limit the present invention to any theory or mechanism, it is believed that pre-apoptotic cells are more susceptible to treatment (e.g., treatment with a hypotonic solution (e.g., ACK buffer)) because they have altered cytoskeletal properties (e.g., undesirable cytoskeletal properties) and altered membrane properties (e.g., undesirable membrane properties), making them more susceptible to rupture. For example, when a treatment (e.g., treatment with a hypotonic solution (e.g., ACK buffer)) is applied to a sample containing a mixed population of pre-apoptotic and non-pre-apoptotic cells, the treatment induces swelling of both populations of cells. However, non-pre-apoptotic cells are able to successfully remodel their membranes and cytoskeleton to compensate for the increase in intracellular fluid. Pre-apoptotic cells have difficulty remodeling their membranes and cytoskeleton, making them more susceptible to rupture when subjected to the treatment.

[0077] Beyond this, and without wishing to limit the invention to any theory or mechanism, it is believed that when a sample containing a mixed population of pre-apoptotic and non-pre-apoptotic cells is subjected to a treatment (e.g., a mechanical treatment such as shear or compression), the non-pre-apoptotic cells are able to successfully remodel their membranes and cytoskeleton to compensate for the mechanical stress. However, pre-apoptotic cells have difficulty remodeling their membranes and cytoskeleton, making the pre-apoptotic cells more susceptible to the treatment applied.

[0078] In some embodiments, the treatment (e.g., the treatment includes a hypotonic solution, e.g., an ACK buffer) reduces pre-apoptotic cells in the sample. In some embodiments, the treatment (e.g., the treatment includes a hypotonic solution, e.g., an ACK buffer) removes a portion of the pre-apoptotic cells in the sample. In other embodiments, the treatment (e.g., the treatment includes a hypotonic solution, e.g., an ACK buffer) reduces pro-apoptotic cells in the sample. In some embodiments, the treatment (e.g., the treatment includes a hypotonic solution, e.g., an ACK buffer) removes a portion of the pro-apoptotic cells in the sample.

[0079] In some embodiments, samples of cells (e.g., chondrocytes, human chondrocytes, non-articular chondrocytes, cells derived from a portion of a rib, etc.) after processing may be used for one or more of the following: direct use of the cells; in vitro culture of the cells, including passaging in a three-dimensional environment, including monolayer or suspension culture; tissue engineering using scaffold-free systems, including self-assembly, or using scaffold-based systems that include natural and synthetic materials; cell transplantation; tissue transplantation; and / or grafting.

[0080] In other embodiments, samples of treated cells (e.g., chondrocytes, human chondrocytes, non-articular chondrocytes, cells derived from a portion of a rib, etc.) or tissues engineered / created from the above samples of generated cells may be further subjected to treatments including one or more of the following: growth factors; cytoskeletal modifying agents; hormones; toxic compounds; molecules acting upstream of signaling cascades; various oxygen tensions; cross-linking agents; matrix degrading enzymes, matrix molecules; and / or mechanical stimuli.

[0081] In some embodiments, the method of preparing a cell population further comprises culturing the population of non-pre-apoptotic cells for the production of neocartilage. In other embodiments, the method of preparing a human cell population further comprises culturing the population of non-pre-apoptotic cells for the production of neocartilage. In some embodiments, the methods described herein can be repeated multiple times, alone or in combination with other treatments.

[0082] The present invention features methods and systems for enhancing cells for therapeutic use, including cell purification methods to enrich cell populations. The cells are used in tissue engineering applications and cell or tissue transplantation. The cell populations can include fully differentiated cells such as chondrocytes, osteoblasts, adipocytes, cardiomyocytes, etc. The tissues can include fat, cartilage, bone, tendon, ligament, muscle, and skin.

[0083] An enriched cell population may be one that has improved homogeneity of cells with properties suitable for cell / tissue engineering, improved robustness of cells, improved cell phenotype, improved tissue engineering, e.g., improved properties that result in faster generation of new tissue or better new tissue constructs.

[0084] The invention features methods that include 1) isolating cells or tissue, e.g., from a donor or source, and 2) chemically or physically / mechanically treating the cells (e.g., chondrocytes). A non-limiting example of chemical treatment includes the introduction of a hypotonic buffer to the cells during cell purification methods, which results in a significantly more mechanically robust neo-tissue construct (e.g., neo-cartilage). The method may include pelleting the cells.

[0085] The present invention features purification methods based on cellular properties including cytoskeleton, membrane surface area, and stiffness. Without wishing to limit the invention to any particular theory or mechanism, it is believed that the purification process preferentially selects for pre-existing cells with undesirable properties or cells with altered phenotypes (compromised cells), including but not limited to fragmented cytoskeleton, reduced membrane surface area, and altered cellular stiffness. These compromised cells are removed to yield a cell population enriched for functional cells and cells with properties that foster the development of new tissue.

[0086] In some embodiments, the cells removed by the process comprise one percent or more of the cartilage-derived cell or tissue population, and the cells removed are designated as having an existing undesirable cytoskeleton, membrane surface area, and / or stiffness. The cell or tissue population used in accordance with the present invention may be cells freshly extracted from cartilage from a living subject, or cells that have been previously frozen or otherwise preserved, or cells that have been previously cultured in vitro or in vivo.

[0087] In some embodiments, cells with pre-existing undesirable cytoskeletal properties include cells with weakened, fragmented, destroyed, or modified cytoskeleton, cells with cytoskeleton that cannot remodel or has reduced remodeling ability, cells with cytoskeletal properties that make the cells more susceptible to destruction by treatment, or combinations thereof. Without wishing to limit the present invention to any particular theory or mechanism, it is believed that at least 1% of a cell population (e.g., a chondrogenic cell population) has pre-existing undesirable cytoskeletal properties. Thus, in some embodiments, treatment using chemical or physical methods (e.g., swelling, shearing, compression) aims to remove at least 1% (but less than 99%) of a cell population (e.g., a chondrogenic cell population) to ensure removal of cells (e.g., chondrocytes) with pre-existing undesirable cytoskeletal properties. For example, screening conditions can be set to cause removal of at least 1% (but less than 99%) of a cell population based on its pre-existing undesirable cytoskeletal properties. In some embodiments, the treatment aims to remove at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75% of the cell population to ensure removal of cells with existing undesirable cytoskeletal properties.

[0088] In some embodiments, cells with undesirable membrane properties include cells with reduced membrane surface area, cells with disrupted or altered membranes, cells whose membranes cannot adapt to conformational changes / size changes, and cells with membrane properties that make the cells more susceptible to destruction by treatment, or combinations thereof. Without wishing to limit the present invention to any particular theory or mechanism, it is believed that at least 1% of a cell population (e.g., a chondrogenic cell population) has pre-existing undesirable membrane surface area properties. Thus, in some embodiments, treatment using chemical or physical methods (e.g., swelling, shearing, compression) aims to remove at least 1% (but less than 99%) of a cell population (e.g., a chondrogenic cell population) to ensure removal of cells (e.g., chondrocytes) with pre-existing undesirable membrane surface area properties. For example, screening conditions can be set to cause removal of at least 1% (but less than 99%) of a cell population based on its pre-existing undesirable membrane surface area properties. In some embodiments, the treatment aims to remove at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75% of the cell population to ensure removal of cells with existing undesirable membrane surface area properties.

[0089] In some embodiments, cells with undesirable stiffness properties include cells with reduced overall stiffness, cells with increased overall stiffness, cells with varying stiffness depending on the region of the cell being tested, cells with reduced flexibility, cells with stiffness that makes the cells more susceptible to destruction by treatment, or combinations thereof. Without wishing to limit the present invention to any particular theory or mechanism, it is believed that at least 1% of a cell population (e.g., a chondrogenic cell population) has pre-existing undesirable stiffness. Thus, in some embodiments, treatment using chemical or physical methods (e.g., swelling, shear, compression) aims to remove at least 1% (but less than 99%) of a cell population (e.g., a chondrogenic cell population) to ensure removal of cells (e.g., chondrocytes) with pre-existing undesirable stiffness. For example, screening conditions can be set to cause removal of at least 1% (but less than 99%) of a cell population based on its pre-existing undesirable stiffness properties. In some embodiments, the treatment aims to remove at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75% of the cell population to ensure removal of cells with existing undesirable stiffness.

[0090] In appropriate circumstances, purification involves subjecting the population of cells to a treatment that: 1) induces cell swelling; 2) induces shear; 3) applies impact or compression, or a combination thereof.

[0091] Non-limiting examples of methods for inducing cell swelling include adding a hypotonic buffer (e.g., ACK buffer) or water, performing a freeze-thaw cycle, reducing dissolved gases, applying a vacuum or negative pressure, or a combination thereof.

[0092] Examples of methods of inducing shear include, but are not limited to, fluid stream shear, opposing microfluidic flow, forcing the cells into small filters / meshes or channels / tunnels, spraying with solutions, or combinations thereof. As used herein, a "small" filter / mesh refers to a filter or mesh smaller than 100 μm. In some embodiments, a small "filter / mesh" refers to a filter or mesh that is about 10 μm to 100 μm, or about 10 μm to 90 μm, or about 10 μm to 80 μm, or about 10 μm to 70 μm, or about 10 μm to 60 μm, or about 10 μm to 50 μm, or about 10 μm to 40 μm, or about 10 μm to 30 μm, or about 10 μm to 20 μm, or about 15 μm to 20 μm, or about 50 μm to 100 μm, or about 50 μm to 90 μm, or about 50 μm to 80 μm, or about 50 μm to 70 μm, or about 50 μm to 60 μm, or about 80 μm to 100 μm, or about 80 μm to 90 μm.

[0093] Non-limiting examples of methods to affect or induce compaction include forcing through small filters / meshes or channels / tunnels, applying mechanical compression, applying physical collisions, or combinations thereof. In some embodiments, the purification method further comprises treating the cells with high frequency vibrations, e.g., by sonication or generating cavitation. In some embodiments, "high frequency" refers to frequencies greater than 10 kHz. In some embodiments, the purification method further comprises adjusting the frequency of the pulse width at about 10 kHz to 100 kHz, or about 10 kHz to 90 kHz, or about 10 kHz to 80 kHz, or about 10 kHz to 70 kHz, or about 10 kHz to 60 kHz, or about 10 kHz to 50 kHz, or about 10 kHz to 45 kHz, or about 10 kHz to about 40 kHz, or about 10 kHz to 35 kHz, or about 10 kHz to 30 kHz, or about 10 kHz to 25 kHz, or about 10 kHz to 20 kHz, or about 10 kHz to 15 kHz, or about 20 kHz to 100 kHz, or about 20 kHz to 90 kHz, or about 20 The present invention also includes treating cells with vibrations of about 20 kHz to 80 kHz, or about 20 kHz to 70 kHz, about 20 kHz to 60 kHz, or about 20 kHz to 50 kHz, or about 20 kHz to 45 kHz, or about 20 kHz to about 40 kHz, or about 20 kHz to 35 kHz, or about 20 kHz to 30 kHz, or about 20 kHz to 25 kHz, or about 50 kHz to 100 kHz, or about 50 kHz to 90 kHz, or about 50 kHz to 80 kHz, or about 50 kHz to 70 kHz, about 50 kHz to 60 kHz, or about 80 kHz to 100 kHz, or about 80 kHz to 90 kHz. In other embodiments, the purification method further comprises treating the cells with a vibration of about 10 kHz, or about 15 kHz, or about 20 kHz, or about 25 kHz, or about 30 kHz, or about 35 kHz, or about 40 kHz, or about 45 kHz, or about 50 kHz, or about 60 kHz, or about 70 kHz, or about 80 kHz, or about 90 kHz, or about 100 kHz. In some embodiments, the purification method further comprises treating the cells with a vibration of greater than 100 kHz.

[0094] In some embodiments, the hypotonic buffer comprises ammonium chloride potassium (ACK) buffer. The ACK buffer may have a composition such as 154 mM ammonium chloride, 10 mM potassium bicarbonate, and 97 μM EDTA. However, the ACK buffer is not limited to this composition. In appropriate situations, the hypotonic buffer comprises Gey buffer, Tris-HCl, HEPES+EGTA+MgCl, MP-40 lysis buffer, RIPA lysis buffer, SDS, hypotonic saline, diluted PBS, purified water, or a combination thereof. The present invention is not limited to the above hypotonic buffer.

[0095] Isolating cells from a donor or source may include obtaining tissue from a donor, digesting the tissue with an enzyme, including collagenase, dispase, pronase, or a combination thereof, filtering the cells from the enzyme-digested tissue, and resuspending the cells in a buffer (e.g., a hypotonic buffer or an alternative buffer) or culture medium.

[0096] Any suitable cell population may be used. For example, the cells may be mammalian cells or plant cells. In some embodiments, the cells include chondrocytes (e.g., primary chondrocytes), osteoblasts, cardiomyocytes, adipocytes, hepatocytes, tenocytes, osteoclasts, smooth muscle cells, pericytes, neurons, fibroblasts, keratinocytes, endothelial cells, muscle cells, mesenchymal stem cells, hematopoietic stem cells, adipose-derived stem cells, or combinations thereof. In some embodiments, the population of cells is a combination of cell types. The present invention is not limited to the above cell types or cell origins.

[0097] In some embodiments, the cells are healthy cells, hi some embodiments, the cells are derived from a diseased tissue or source (e.g., osteoarthritic cartilage).

[0098] The method of the present invention further includes introducing a cytoskeleton modifier, an actin polymerization inhibitor (e.g., cytochalasin D), and / or a cytoskeleton polymerization modifier (e.g., an inhibitor or enhancer, e.g., an inhibitor of microtubule polymerization) into the cells already purified by the above hypotonic buffer. The cytoskeleton modifier and / or the actin polymerization inhibitor and / or the cytoskeleton polymerization modifier can further enhance the mechanical properties and matrix deposition of the cells. The present invention is not limited to cytochalasin D.

[0099] In some embodiments, the cytoskeletal modifiers and / or actin polymerization inhibitors and / or cytoskeletal polymerization modifiers include microfilament or actin stabilizers, polymerization agents or inhibitors (e.g., cytochalasin family, alternative cytochalasins, latrunculins, jasrakinolides, phalloidins, swinholides, colchicine), intermediate filament stabilizers, polymerization agents or inhibitors, microtubule stabilizers, polymerization agents or inhibitors, lysophosphatidic acid, staurosporine, blebbistatin, Y27632, septins, and combinations thereof. These agents (cytoskeletal modifiers and / or actin polymerization inhibitors and / or cytoskeletal polymerization modifiers) are compounds that act directly or indirectly on the cytoskeleton (e.g., Y27632, which acts upstream of the signaling cascade to affect myosin function). As a non-limiting example, the addition of cytochalasin D can improve the mechanical properties and matrix deposition of neocartilage engineered with chondrocytes purified in hypotonic buffers and passaged multiple times. The present invention is not limited to the above compounds.

[0100] The method may further include treating the cells with a cytoskeletal modifying agent, an actin polymerization inhibitor (eg, cytochalasin D), a cytoskeletal polymerization modifying agent, or a combination thereof, prior to treating the cells with the hypotonic buffer.

[0101] In some embodiments, the cytoskeletal modifying agent, actin polymerization inhibitor, or cytoskeletal polymerization modifying agent acts directly or indirectly upstream of a signaling cascade. The cytoskeletal modifying agent inhibits, stabilizes, or strengthens the cytoskeleton.

[0102] In some embodiments, cytochalasin D (or a cytoskeletal modifier, actin polymerization inhibitor, and / or a cytoskeletal polymerization modifier) ​​is used from 0 to 48 hours during neocartilage formation.

[0103] In some embodiments, the hypotonic buffer is introduced after cell isolation from tissue, after thawing, after monolayer growth, after redifferentiation, or before new tissue formation. The hypotonic buffer can be applied to the tissue using mechanical means or perfusion.

[0104] The method of treating a subject may involve using the isolated and maintained cells directly for therapy.

[0105] The method may further comprise subjecting the isolated and maintained cells to two-dimensional culture with monolayer passaging to any extent.

[0106] The method may further comprise subjecting the isolated, retained cells to three-dimensional culture, including one or more of the following: 1) suspension culture; 2) with scaffolds of any shape or size, such as hydrogels, collagen gels, alginates, decellularized membranes or tissues, dehydrated membranes or tissues, lyophilized membranes or tissues, ceramics, such as hydroxyapatite of all stoichiometries, alpha-tricalcium phosphate, beta-tricalcium phosphate, natural matrices, such as silk, synthetic materials, such as poly(lactic acid) or polylactide (PLA), poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), polyglycolide (PGA), polycaprolactone, or combinations thereof; 3) scaffold-free techniques, such as self-assembly, pellet culture, aggregate culture, cell sheets, tissue fusion, or any combination thereof; 4) scaffold-free and scaffold-based combinations; 5) alone or with other types and treatments of cells.

[0107] The method may further include seeding the isolated, retained cells (e.g., after pelleting). The cells may be seeded into a non-adherent well. The method may further include seeding cells (e.g., chondrocytes) into the non-adherent well, e.g., after pelleting, wherein the cells seeded into the non-adherent well form neo-cartilage. The invention is not limited to seeding cells into a non-adherent well.

[0108] In some embodiments, the resulting neo-cartilage has increased mechanical properties (e.g., one or more of aggregate modulus, shear modulus, tensile modulus, compressive stiffness, tensile stiffness, and tensile strength) compared to neo-cartilage made from chondrocytes that have not been treated with a hypotonic buffer (e.g., ACK buffer). In other embodiments, the resulting neo-cartilage has a correct morphology (e.g., flat and not curled) compared to neo-cartilage made from chondrocytes that have not been treated with a hypotonic buffer (e.g., ACK buffer).

[0109] In some embodiments, the neo-cartilage may have improved synthesis and deposition of neo-cartilage matrix compared to neo-cartilage generated from chondrocytes that have not been treated with a hypotonic buffer, hi some embodiments, the neo-cartilage may have improved collagen cross-linking compared to neo-cartilage generated from chondrocytes that have not been treated with a hypotonic buffer.

[0110] In some embodiments, the donor is a fetal donor, a juvenile donor, or an adult donor.

[0111] The method may further include subjecting the isolated and retained cells to a chemical factor or a bioactive agent, non-limiting examples of which include active and latent growth factors (e.g., TGF superfamily, growth differentiation factors, bone morphogenetic proteins), cytoskeletal modifiers (cytochalasin D), bioactive agents, hormones (e.g., triiodothyronine, parathyroid hormone), mitogens, enzymes (e.g., chondroitinase-ABC, lysyl oxidase, lysyl oxidase, lysyl oxidase-like 2), collagen cross-linking agents, toxic compounds, molecules acting upstream in a signaling cascade, or combinations thereof.

[0112] The method may further include subjecting the isolated, retained cells to one or more of SZP / PRG4, chondroitin sulfate, linking protein, hyaluronan, keratin sulfate, dermatan sulfate, and aggrecan, molecules including collagen types I, II, III, V, VI, X, and XI, or any agent that increases production of these molecules.

[0113] The method may further include subjecting the isolated, retained cells to different oxygen tensions achieved by environmental oxygen deficiency or enzymatic conditions.

[0114] The method can further include treating the cells with a physical stimulus, for example, static or dynamic direct compression, hydrostatic pressure, shear, tension, fluid flow induced shear, perfusion, or a combination thereof.

[0115] The method may further include treating the isolated and retained cells with hyaluronidase in combination with a cytoskeletal modifying agent, an actin polymerization inhibitor, or a cytoskeletal polymerization modifying agent.

[0116] The methods of the invention enrich cell populations. The methods may improve cell homogeneity. The methods may improve the robustness of cell populations.

[0117] The methods may further include using the isolated and maintained cells in combination with other prepared cells and tissues.

[0118] The method may be used on cells or tissues for the purposes of tissue engineering, e.g. cartilage tissue engineering. The method may be used on cells or tissues for the purposes of cell transplantation, e.g. autologous chondrocyte implantation (ACI). The method may be used on tissues for the purposes of tissue transplantation, e.g. mosaicoplasty.

[0119] Without wishing to limit the invention to any theory or mechanism, it is believed that the methods and systems of the present invention can improve the mechanical properties of new tissues generated from fetal age cells to those generated from adult level cells. Without wishing to limit the invention to any theory or mechanism, it is believed that the methods and systems of the present invention are advantageous since there are currently no standardized methods for chondrocyte purification.

[0120] The present invention is not limited to cells used for engineering applications. For example, the methods and systems of the present invention can be used for a variety of different applications, such as cancer cell applications, cell purification methods, transplantation (e.g., fat transplantation). In some embodiments, the present methods of enriching cell populations provide a desired cell population for use before or in preparation for treating a subject. The enriched cells can be administered directly to a subject (enriched use). The enriched cells can be further cultured in two-dimensional in vitro, including passaging in monolayers, before administration to a subject (enriched use). The enriched cells can be further cultured in three-dimensional in vitro, including suspension culture, before administration to a subject (enriched use). The enriched cells can be further cultured in vitro for tissue engineering, using a scaffold-free system, including self-assembly, or using a scaffold-based system, including natural and synthetic materials, before administration to a subject (enriched use). The enriched cells can be used for cell transplantation, tissue transplantation, and / or transplantation to treat a subject (enriched use). One or more of these enriched uses can be performed after the enrichment method.

[0121] The present invention is not limited to cells used in engineering applications, for example, the methods and systems of the present invention may be used in a variety of different applications, such as cancer cell applications, cell purification methods, transplantation (e.g., fat transplantation).

[0122] The hypotonic buffer can be introduced at any time during culture, such as after monolayer growth, after redifferentiation, or before neotissue formation, to generate an enriched cell population that is free of cells with pre-existing undesirable cytoskeleton, membrane surface area, and stiffness properties. As previously mentioned, the present invention is not limited to ACK buffer.

[0123] As mentioned above, cytoskeleton modifiers and / or actin polymerization inhibitors (e.g., cytochalasin D) and / or cytoskeleton polymerization modifiers may be used in some cases. Example 4 below describes the use of cytochalasin D. As an example, in some embodiments, 2 μM cytochalasin D may be used at 0-48 hours during neocartilage formation via the self-assembly method. It should be noted that the present invention is not limited to Example 4. Cytochalasin D may be used in other cartilage tissue engineering systems, such as, but not limited to, self-assembly or scaffold-based systems, as well as other sources of chondrocytes, such as nasal or aural chondrocytes or osteoarthritic chondrocytes.

[0124] The methods described herein can be used independently or in combination. Purification treatments (e.g., hypotonic buffers) and / or the use of cytoskeletal modifying agents can be applied at various times throughout the culture.

[0125] The invention also features tissue engineering of various tissues such as articular cartilage, or cell transplantation, or fat grafting using purified cells. In some embodiments, the pelleted cells are for cell transplantation, or tissue engineering, or for transplantation. In some embodiments, the pelleted cells are for cell injection.

[0126] In some embodiments, the method of the present invention includes isolating cells from a donor; treating the cells with a hypotonic buffer; pelleting the cells; passaging / growing the cells in a monolayer, redifferentiating the cells, and seeding the redifferentiated cells. The cells can be seeded in a non-adherent well (e.g., a non-adherent agarose well). The present invention is not limited to seeding cells in a non-adherent well. The techniques for tissue engineering can be scaffold-based or scaffold-free.

[0127] In some embodiments, the methods of the invention are for preparing new tissue made from fetal age chondrocytes whose mechanical properties are similar to those of adult articular cartilage.

[0128] The methods may be for enriching populations of functional cells and / or cells with pre-existing properties conducive to neotissue formation, including but not limited to cells with an intact cytoskeleton that can be remodeled, cells with high membrane surface area, and cells with unchanged stiffness (cells capable of undergoing conformational changes). The methods may be for enriching populations of cells to engineer native-like neocartilage. The methods may be for enriching populations of cells to engineer native-like neotissue.

[0129] In some embodiments, the methods of the invention allow for the use of lower seeding densities (e.g., for neotissue generation), e.g., the methods of the invention improve the robustness of the cell population, such that fewer cells are required (e.g., compared to other methods). In some embodiments, a seeding density of about 2 million cells per construct is used. In some embodiments, using a seeding density of about 2 million cells per construct further increases the aggregate modulus and shear modulus.

[0130] It is noted that in the present invention, additional biochemical treatments and / or mechanical stimuli may be used in combination with (i) a hypotonic buffer; (ii) a cytoskeletal modifier, an actin polymerization inhibitor (e.g., cytochalasin D), a cytoskeletal polymerization modifier, or a combination thereof; or (iii) both a hypotonic buffer and a cytoskeletal modifier, an actin polymerization inhibitor (e.g., cytochalasin D), a cytoskeletal polymerization modifier, or a combination thereof. For example, the present invention relates to: (A) the use of a hypotonic buffer to prepare cells for cell transplantation and / or tissue engineering in scaffold-free or scaffold-based systems ((i) the preparation may include the use of a physical stimulus (e.g., shear), (ii) the preparation may feature additional treatment by biochemical treatment, (iii) the preparation may feature additional stimulation by mechanical means, and (iv) the preparation may feature additional treatment and stimulation by biochemical and mechanical means); (B) the use of a cytochalasin to reinforce engineered neocartilage (both scaffold-free and scaffold-based systems). (i) the preparation may be characterized by additional treatment by biochemical treatment; (ii) the preparation may be characterized by additional stimulation by mechanical means; (iii) the preparation may be characterized by additional treatment and stimulation by biochemical and mechanical means); and (C) the use of a hypotonic buffer and cytochalasin D ((i) the preparation may be characterized by additional treatment by biochemical treatment; (ii) the preparation may be characterized by additional stimulation by mechanical means; (iii) the preparation may be characterized by additional treatment and stimulation by biochemical and mechanical means).

[0131] In summary, non-limiting examples of the present invention include: (1) hypotonic buffer; (2) cytochalasin D; (3) hypotonic buffer + cytochalasin D; (4) hypotonic buffer + biochemical treatment; (5) hypotonic buffer + physical stimulation; (6) hypotonic buffer + biochemical treatment + physical stimulation; (7) cytochalasin D + biochemical treatment; (8) cytochalasin D + physical stimulation; (9) cytochalasin D + biochemical treatment + physical stimulation; (10) hypotonic buffer + cytochalasin D + biochemical treatment; (11) hypotonic buffer + cytochalasin D + physical stimulation; (12) hypotonic buffer + cytochalasin D + biochemical treatment + physical stimulation. Note that the above cytochalasin D may be replaced by a cytoskeleton modifier, an actin polymerization inhibitor, a cytoskeleton polymerization modifier, or a combination thereof.

[0132] The methods and systems of the invention (e.g., use of hypotonic buffers, use of cytoskeletal modifiers and / or actin polymerization inhibitors and / or cytoskeletal polymerization modifiers) can be used independently or in combination with each other, or in combination with other bioactive agents (e.g., growth factors, chondroitinase ABC, lysyl oxidase-like 2) and physical / mechanical stimuli (e.g., direct compression, shear, hydrostatic pressure, tension) to achieve, for example, greater functional properties of engineered neo-tissue (e.g., articular cartilage).

[0133] Without wishing to limit the present invention to any theory or mechanism, it is believed that treatment with cytoskeletal modifiers and / or actin polymerization inhibitors (e.g., cytochalasin D) and / or cytoskeletal polymerization modifiers is advantageous as it helps induce native-like compressibility in engineered neocartilage. In particular, multiple passages of fetal chondrocytes treated with cytochalasin D while undergoing self-assembly formed neocartilage with compressibility comparable to native adult cartilage. This level of mechanical robustness has not been seen before with a fetal chondrocyte source.

[0134] The following example describes the use of ACK buffer on chondrocyte isolation from bovine fetal and bovine juvenile sources, which resulted in significant improvements in homogeneity, matrix deposition, and mechanical properties of neocartilage constructs.

[0135] For example, without wishing to limit the invention to any theory or mechanism, it is believed that during a biopsy to obtain a sample of chondrocytes, cells from the surrounding tissue or hematopoietic cells (e.g., pro-apoptotic cells) may be contaminating the sample. Exposure of chondrocytes to hematopoietic cells (e.g., pro-apoptotic cells) during or after collection of the cartilage sample can render the chondrocytes pre-apoptotic or apoptotic.

[0136] Without wishing to limit the invention to any particular theory or mechanism, it is believed that the purification methods are effective in increasing the functionality of therapeutic cells by reducing contaminating cells (e.g., pre-apoptotic or pro-apoptotic cells), particularly by reducing populations of cells that have existing undesirable properties of compromised cells, including, but not limited to, cells with weakened cytoskeleton, low membrane surface area, and high stiffness.

[0137] Certain embodiments herein, such as the methods herein, may include obtaining a sample of chondrocytes. In some embodiments, the sample of chondrocytes comprises a mixed population of pre-apoptotic chondrocytes and non-pre-apoptotic chondrocytes. In some embodiments, the method includes treating the cells with a hypotonic solution and selectively removing the pre-apoptotic chondrocytes. In some embodiments, the sample of chondrocytes after treatment with the hypotonic solution is more homogenous.

[0138] Certain embodiments herein, such as the methods herein, may include obtaining a sample of cells sourced from a portion of a rib. In some embodiments, the sample of cells includes a mixed population of non-pre-apoptotic cells and pre-apoptotic cells. In some embodiments, the method includes treating the cells with a hypotonic solution and selectively removing the pre-apoptotic cells. In some embodiments, the sample of cells after treatment with the hypotonic solution is more homogenous.

[0139] Certain embodiments herein, such as the methods herein, may include obtaining a sample of human cells sourced from a portion of a rib. In some embodiments, the sample of human cells comprises a mixed population of non-pre-apoptotic human cells and pre-apoptotic human cells. In some embodiments, the method includes treating the cells with a hypotonic solution and selectively removing the pre-apoptotic human cells. In some embodiments, the sample of human cells after treatment with the hypotonic solution is more homogenous.

[0140] The invention may feature a method of preparing a cell sample, the method comprising obtaining a chondrocyte sample comprising a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes, and subjecting the chondrocyte sample from (a) to a hypotonic solution that reduces pre-apoptotic cells in the sample. In some embodiments, the method can be repeated multiple times, alone or in combination with other treatments. In some embodiments, the hypotonic solution is ammonium chloride potassium lysis buffer (ACK buffer). After (b), the chondrocyte sample has a higher percentage of non-pre-apoptotic cells compared to the chondrocyte sample before (b).

[0141] The invention may also feature a method of preparing a cell preparation, the method including obtaining a sample of chondrocytes and subjecting the sample of chondrocytes to a hypotonic solution. In some embodiments, the hypotonic solution is ammonium chloride potassium lysis buffer (ACK buffer). In some embodiments, the cell preparation has a higher percentage of non-pre-apoptotic cells compared to the sample of chondrocytes originally obtained.

[0142] The invention may further feature a method of preparing a cell preparation, comprising obtaining a sample of chondrocytes and subjecting the sample of fully differentiated chondrocytes to a hypotonic solution. In some embodiments, the hypotonic solution is ammonium chloride potassium lysis buffer (ACK buffer). The method enhances the ability of the sample to form a biofunctional tissue.

[0143] The invention further features a method of preparing a sample of human chondrocytes. The method may include obtaining a sample of human chondrocytes sourced from a portion of a rib and subjecting the sample of human chondrocytes to treatment with a hypotonic solution (e.g., ammonium chloride potassium lysis (ACK) buffer). The method may further include cutting the portion of the rib before subjecting the sample of human chondrocytes to treatment. In some embodiments, cutting the portion of the rib can remove cells that are not suitable for tissue formation (e.g., remove undesirable cell types). The method may further include enzymatically digesting the portion of the rib before subjecting the sample of human chondrocytes to treatment. In some embodiments, the sample of chondrocytes is a primary cell sample.

[0144] The invention may also feature a treated cell sample produced from a method including (a) obtaining a sample of cartilage and digesting the cartilage sample to obtain a cell suspension; and (b) subjecting the cell suspension to a hypotonic solution to obtain a treated cell sample. In some embodiments, the treated cell sample is suitable for neocartilage production. In some embodiments, the sample of chondrocytes is a primary cell sample. The method may further include, after (b), centrifuging the treated cell sample and resuspending the cell sample.

[0145] The present invention is not limited to the methods or compositions described herein.

[0146] example The following are non-limiting examples of the present invention. It should be understood that the above examples are not intended to limit the present invention in any way. Equivalents or alternatives are within the scope of the present invention.

[0147] A. Purification based on cytoskeletal properties Example 1 - Hypotonic solution Example 1 describes a method to select cells based on cytoskeletal properties using a hypotonic solution. Example 1 shows that treatment of freshly isolated, fully differentiated cells with ACK buffer, a hypotonic solution, enhances their ability to form biofunctional tissue. Clinically relevant articular chondrocytes (AC) derived from fetal and juvenile cartilage were used as models in the following studies: fetal ovine articular chondrocytes (foAC) were treated with ACK buffer during their isolation. Without wishing to attribute the invention to any particular theory or mechanism, it is believed that treatment of chondrocytes with a hypotonic buffer is effective in increasing viable chondrocyte purity by reducing the number of cells with existing undesirable cytoskeletal properties. Thus, this treatment increases the functional properties of the resulting self-assembling neocartilage by generating a population of cells enriched for viable chondrocytes without undesirable cytoskeletal properties. The effect of ACK buffer treatment on cells derived from animal models of different species and ages, in particular juvenile bovine articular chondrocytes (jbAC), was examined.

[0148] Cell isolation: foAC was harvested from the patellofemoral surface of the stifle joint of three fetal (120-125 days gestation), female, Dorpa-cross sheep. jbAC was harvested from the patellofemoral surface of the stifle joint of three juvenile (2-14 days), male, Holstein and Jersey calves. Processing of ovine and bovine tissue was identical. Articular cartilage from the entire surface of both the condyle and trochlear groove was extracted from approximately 1 mm 3 After chopping into pieces, the tissues were washed and centrifuged (500G, 5 min) three times with Dulbecco's modified Eagle's medium containing 4.5 g / L glucose and GlutaMAX (DMEM; Gibco) and 2% (v / v) penicillin / streptomycin / fungizone (PSF; BD Biosciences). The tissues were digested in 0.2% (w / v) collagenase type II (Worthington) in DMEM containing 3% (v / v) fetal bovine serum (FBS; Atlanta Biologicals) for 18 hours at 37°C with gentle rocking. After digestion, the resulting cell solution was filtered through a 70 μm cell strainer, centrifuged (500G, 5 min), and resuspended in blank DMEM. ACs and RBCs were counted, and AC viability was assessed by trypan blue staining. Half of the foACs and half of the jbACs were treated with ACK buffer, as described in detail below. Cells were counted and viability was assessed again after ACK buffer treatment. Untreated cells were washed with blank DMEM instead of ACK buffer but otherwise handled in the same manner. Cells immediately underwent self-assembly.

[0149] ACK Buffer Treatment: ACK buffer consisted of 154.4 mM ammonium chloride (Sigma), 10 mM potassium bicarbonate (Sigma-Aldrich), 97.3 μM ethylenediaminetetraacetic acid (EDTA) tetrasodium salt (Acros Organics). This corresponds to 8.26 g ammonium chloride, 1.0 g potassium bicarbonate, and 0.037 g EDTA in 1 L of ultrapure water. The solution was sterile filtered before use.

[0150] Protocol for purifying chondrocytes by introducing ACK buffer: (1) Warm ACK buffer to 37°C. (2) Place up to 100 million chondrocytes in a 50mL conical tube. (3) Centrifuge the cell solution at 500G for 5 minutes. (4) Aspirate the supernatant and gently resuspend the cell pellet in 10mL ACK buffer. Incubate at 37°C for 3-5 minutes. (5) Centrifuge the ACK buffer cell suspension at 500G for 5 minutes. (6) Aspirate the ACK buffer. Wash the cell pellet twice with blank or wash medium before plating or freezing.

[0151] Seeding and culture of neocartilage constructs: Primary foAC and jbAC treated with ACK buffer (+ACK-treated) and untreated (-ACK-treated) were each allowed to self-assemble into engineered neocartilage constructs in non-adherent agarose wells. Sterile stainless steel molds consisting of 5 mm diameter cylindrical posts were inserted into a 48-well plate to create a single agarose well within each plate well, with each well containing 1 mL of molten 2% (w / v) molecular biology grade agarose (Thermo). After allowing the agarose to solidify at room temperature, the molds were removed. Agarose wells were filled with chemically defined chondrogenic medium (CHG medium) (DMEM containing 1% PSF, 1% ITS+premix (BD Biosciences), 1% non-essential amino acids (Gibco), 100 nM dexamethasone (Sigma), 50 mg / mL ascorbate-2-phosphate (Sigma), 40 g / mL L-proline (Sigma), and 100 mg / mL sodium pyruvate (Sigma)). CHG medium was changed twice over a 5-day period to ensure saturation of the agarose prior to cell seeding. Treated and untreated foAC and jbAC were each seeded into 5 mm agarose wells at 4.5 million cells in 100 μL of CHG medium per construct. Constructs were unconfined on day 6 and placed into larger wells that were coated with agarose to prevent adhesion of the constructs to the wells. The medium was changed daily before release and every other day after the 6-week culture period. Gross morphological analysis, histology, immunohistochemistry (IHC), glycosaminoglycan (GAG) and collagen quantification, and mechanical evaluation were performed at the end of the culture period.

[0152] Gross morphological analysis: Construct thickness was measured from construct photographs using ImageJ software (National Institutes of Health). Entire constructs were weighed to obtain wet weights, and samples were then sectioned for histological, biochemical, and mechanical analysis.

[0153] Histological and immunohistochemical (IHC) evaluation: Samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned into 5 μm sections along the short axis to expose the full thickness of the construct. Sections were stained with Hematoxylin and Eosin (H&E) to show morphology, Safranin O / Fast Green to visualize GAGs, and Picrosirius Red to visualize collagen. In addition, IHC was performed for collagen I (ab90395, dilution 1:250, Abcam) and collagen II (ab34712, dilution 1:4000, Abcam).

[0154] Biochemical evaluation: Construct samples aliquoted for biochemical analysis were weighed to determine wet weight, lyophilized, and weighed again to determine dry weight. Construct hydration was performed by normalizing the difference in weight before and after lyophilization to the wet weight of the sample. Lyophilized samples were digested in 125 μg / mL papain (Sigma-Aldrich) at 65° C. for 18 hours. GAG content was quantified by Blyscan assay kit (Biocolor). Collagen content was quantified by a modified colorimetric chloramine-T hydroxyproline assay. A standard curve was generated using Sircol collagen standards (Biocolor). DNA content was quantified by PicoGreen dsDNA reagent (Invitrogen). Both collagen and GAG content were normalized to wet weight, dry weight, and DNA content.

[0155] Mechanical evaluation: Creep indentation compression tests were performed on 3 mm diameter punches from each construct. A flat porous indenter tip with a diameter of 0.8 mm was applied to the samples with masses ranging from 0.45 to 2 g to achieve strains of 10-15%. Aggregate and shear moduli were obtained from experimental data using semi-analytical, semi-numerical, linear biphasic, and finite element models. For tensile testing, samples were punched into dog-bone shaped specimens with a gauge length of 1.92 mm according to ASTM standards (ASTM D3039). Paper tabs were attached to the samples outside the gauge length and gripped in a TestResources machine (TestResources Inc.) and pulled at 1% of the gauge length per second until the samples broke. The cross-sectional area of ​​the samples was measured by ImageJ and used to generate stress-strain curves. Tensile modulus was obtained by least squares fit of the linear region of the curve. The maximum stress gave the ultimate tensile strength (UTS).

[0156] Statistical analysis: Biochemical and mechanical data were analyzed using Student's t-test in Prism 6 (GraphPad Software). A p-value of <0.05 indicates statistical significance. A sample size of n=6 per group was used. In figures showing quantitative results, groups not shown with the same symbol are statistically different. All data are presented as mean ± standard deviation.

[0157] Results: Figure 1 shows the isolated cell pellet morphology and cell counts before and immediately after ACK buffer treatment. ACK buffer treatment resulted in morphological changes in the pellets of both cell types. The foAC pellets before treatment appeared pale red throughout treatment and milky white after treatment. The jbAC pellets appeared tan with a pink cast before treatment and milky white after treatment. The viability of foAC before and after treatment was 84±11% and 82±7%, respectively. The viability of jbAC before treatment was 92±7%, and the viability of jbAC after treatment was 86±3%. The total numbers of foAC and jbAC were reduced by 19±7% and 9±3%, respectively, with ACK treatment. The RBC content was significantly reduced after treatment for both foAC (36±14% before treatment and 14±3% after treatment) and jbAC (21±6% before treatment and 7±2% after treatment).

[0158] FIG. 2 shows the self-assembled neocartilage constructs after 6 weeks of culture. All constructs appeared hyaline-like with similar diameters. Bulbous diffuse areas (indicating areas where "bad" cells were unable to make functional cartilage; the "bad" cells may have exhibited fragmented / inactive cytoskeleton, reduced membrane surface area, and / or altered cell stiffness) were present in both foAC and jbAC untreated groups. ACK treatment eliminated these areas, resulting in flat foAC and jbAC neocartilage. ACK treatment also reduced the thickness and wet weight of both foAC and jbAC neocartilage constructs. The thickness of foAC neocartilage was 1.2±0.1 mm without treatment and was significantly reduced to 0.7±0.1 mm with treatment. The thickness of jbAC neocartilage was 0.58±0.1 mm without treatment and was significantly reduced to 0.38±0.1 mm with treatment. The wet weight of foAC neocartilage was 26.6 ± 0.8 mg without treatment and was significantly reduced to 15.1 ± 0.6 mg with treatment. The wet weight of jbAC neocartilage was 13.3 ± 0.4 mg without treatment and was significantly reduced to 7.3 ± 0.2 mg with treatment. The hydration of foAC neocartilage was 87.1 ± 0.5% without ACK treatment and 87.2 ± 0.4% with treatment. The hydration of jbAC neocartilage was 89.0 ± 0.3% without ACK treatment and was significantly reduced to 86.4 ± 0.9% with treatment.

[0159] FIG. 3 shows the histology and immunohistochemistry of the neocartilage constructs after 6 weeks of culture. Histology showed the presence of diffuse GAG-rich regions with low cellularity in both untreated foAC and jbAC neocartilage. ACK treatment eliminated these diffuse regions resulting in stronger uniform tissue staining for GAG and collagen in both foAC and jbAC constructs. Strong GAG staining was present across all groups, which was further increased by ACK treatment for both foAC and jbAC constructs. Collagen staining was present across all groups, but was additionally enhanced by ACK treatment for both foAC and jbAC constructs. Collagen I staining was absent in either untreated or treated foAC and jbAC constructs. Collagen II staining was present in both untreated foAC and jbAC constructs and was enhanced by ACK treatment.

[0160] Figure 4 shows the biochemical content of the neocartilage constructs. Untreated and ACK-treated foAC neocartilage GAG / wet weight (GAG / WW) were 5.5±0.1% and 5.7±0.2%, respectively. Untreated and ACK-treated foAC neocartilage GAG / dry weight (GAG / DW) were 42.8±1.5% and 43.1±1.7%, respectively. GAG per DNA in untreated foAC constructs was 60.4±0.9 μg / μg and was significantly reduced to 50.54±1.3 μg / μg by ACK treatment. ACK treatment significantly reduced jbAC construct GAG per wet weight from 3.9±0.2% to 3.0±0.1% and GAG per dry weight from 33.5±2.0% to 24.8±2.8%. ACK treatment significantly reduced jbAC construct GAG per DNA from 70.65 ± 5.3 μg / μg to 28.1 ± 1.4 μg / μg.

[0161] Collagen content per wet weight (collagen / WW) and per dry weight (collagen / DW) in foAC neocartilage was significantly increased by ACK treatment from 2.0±0.1% to 2.3±0.1% and from 14.4±0.8% to 18.5±0.7%, respectively. Construct collagen per DNA in untreated and ACK-treated foAC neocartilage was 20.5±0.9μg / μg and 20.4±0.8μg / μg, respectively. ACK treatment significantly increased collagen per wet weight from 1.8±0.1% to 2.0±0.1% in jbAC constructs. Collagen per dry weight in untreated jbAC constructs was 15.2±0.5% and 16.3±1.4% in ACK-treated constructs. Collagen per DNA in the untreated jbAC construct was 31.7±1.2 μg / μg and was significantly reduced to 18.6±0.7 μg / μg by ACK treatment.

[0162] FIG. 5 shows the mechanical properties of the neo-cartilage constructs. ACK treatment significantly enhanced the compressive, shear, and tensile properties of both foAC and jbAC neo-cartilage constructs. The aggregate modulus of the foAC constructs was significantly increased by ACK treatment from 37.8±8.1 kPa to 104.5±13.5 kPa. ACK treatment similarly and significantly increased the aggregate modulus of the jbAC constructs from 83.8±7.0 kPa to 116.6±8.8 kPa. The shear modulus of the foAC and jbAC neo-cartilage was significantly increased by ACK treatment from 21.6±3.5 kPa to 49.4±6.4 kPa and from 38.5±3.3 kPa to 51.9±4.0 kPa, respectively. ACK treatment significantly increased the tensile modulus of the foAC constructs from 0.8±0.1 MPa to 1.5±0.1 MPa and the ultimate tensile strength (UTS) from 0.2±0.1 MPa to 0.5±0.1 MPa. As a result of ACK treatment, the tensile modulus of the jbAC constructs increased significantly from 1.2±0.1 MPa to 1.8±0.1 MPa and the UTS increased significantly from 0.6±0.1 MPa to 1.1±0.1 MPa.

[0163] Example 2 - Shear Example 2 describes a method to select cells based on cytoskeletal properties using shear. Example 2 shows a protocol for purifying articular chondrocytes using shear.

[0164] Cell isolation: Juvenile ovine articular chondrocytes (joAC) are isolated from the femoral condyles and trochlear groove of juvenile Rambouillet Suffolk cross sheep obtained from a local slaughterhouse (Nature's Bounty Farms, Dixon, Calif.) on the same day of animal sacrifice. Cartilage is isolated from 1-2 mm 3 The cartilage is chopped into cubes and washed twice with washing medium (Dubelco's modified Eagle's medium; DMEM containing 1% (v / v) PSF). The chopped cartilage is digested with 500 units / mL collagenase type 2 (Worthington Biochemical) in chondrogenic medium + 3% (v / v) fetal bovine serum (FBS; Atlanta Biologicals) for 18 hours at 37°C and 10% CO2 on an orbital shaker. The cells are then strained through a 70 μm strainer and counted.

[0165] Protocol for introducing shear to purify chondrocytes: (1) Place approximately 30 mL of cell solution into a conical tube. (2) Attach a conical tube filter containing a mesh size of 15-20 μm so that the vacuum pushes the flow of cell solution into a new conical tube. (3) Attach a new conical tube opposite the vacuum filter and attach the filter to the vacuum line. (4) Invert the conical tube and filter setup to allow the cell solution to flow through the filter into the new conical tube. Wait until all the solution has passed through the filter. (5) Remove the filter and old conical tube. Wash the filtered cell solution twice with wash medium and count the remaining cells.

[0166] Example 3 - Impact / Compression Example 3 describes a method to select cells based on cytoskeletal properties using impact / compression. Example 3 shows a protocol for purifying articular chondrocytes using compression / impact.

[0167] Cell isolation: Juvenile ovine articular chondrocytes (joAC) are isolated from the patellofemoral surface of 1-year-old Rambouillet Suffolk cross sheep obtained from a local slaughterhouse (Superior Farms, Dixon, CA) within 48 hours of slaughter (n=8). Cartilage from both the condylar and trochlear surfaces is minced into approximately 1 mm3 pieces and washed three times with Dulbecco's modified Eagle's medium containing 4.5 g / L glucose and GlutaMAX (DMEM; Gibco) and 2% (v / v) penicillin / streptomycin / fungizone (PSF; Lonza). Cartilage is then digested in 0.2% (w / v) collagenase type II (Worthington) in DMEM containing 3% (v / v) fetal bovine serum (FBS; Atlanta Biologicals) for 18 hours at 37°C with gentle rocking. After digestion, the resulting cell solution is filtered through a 70 μm cell strainer.

[0168] Protocol for introducing compression / impact to purify chondrocytes: (1) Place approximately 30 mL of cell solution into a conical tube. (2) Add five glass beads with diameters of 0.5-1.25 mm to the tube. (3) Gently rotate the conical tube on a plate rocker for 3 minutes. (4) Pipette the cell solution into a new conical tube. Wash the glass beads three times with washing medium and place these washing solutions into a new conical tube as well. (5) Wash the processed cell solution twice with washing medium and count the remaining cells.

[0169] B. Purification based on membrane surface area properties Example 4 - Hypotonic solution Example 4 describes a method to select cells based on membrane surface area properties using a hypotonic solution. Example 4 shows that native-like neocartilage is achieved using multiple passaged chondrocytes. The present invention is not limited to the methods or compositions described herein. Example 4 used a cartilage engineering model of the self-assembly method. Without wishing to limit the present invention to any theory or mechanism, it is believed that treatment of primary chondrocytes with a hypotonic buffer is effective in increasing viable chondrocyte purity by reducing the population of cells with pre-existing undesirable membrane surface area properties. This treatment is then believed to increase the functional properties of the resulting self-assembling neocartilage by generating a cell population enriched for viable chondrocytes without pre-existing undesirable membrane surface area properties.

[0170] Example 4 shows that mimicking cell proliferation (chondrogenically regulated proliferation), condensation, differentiation (aggregate redifferentiation culture), cartilage matrix production (self-assembly), and matrix maturation in vitro (using chondrocytes purified with hypotonic solutions and then extensively passaged) results in neocartilage with mechanical properties equivalent to the native articular cartilage from which the cells were sourced. Example 4 describes three phases. In phase 1, the seeding density was determined for both primary and passaged / redifferentiated chondrocytes, e.g., the seeding density that results in neocartilage constructs with the greatest functional properties (and to select a culture system that requires the fewest number of chondrocytes). Without wishing to limit the invention to any theory or mechanism, it is believed that mimicking the developmental sequence of chondrogenically regulated cell proliferation, condensation, and aggregate redifferentiation under optimized culture conditions results in neocartilage derived from purified, multi-passage cells that is equivalent to neocartilage derived from primary cells. In phase 2, the utility of cytochalasin D and hyaluronidase treatment to further promote chondrogenic redifferentiation of expanded chondrocytes was determined. Without wishing to limit the invention to any theory or mechanism, it is believed that the combined treatment promotes cartilage-specific matrix production to increase the functional properties of the neo-cartilage construct. Phase 3 promoted matrix formation and cross-link-based maturation in the neo-cartilage. Without wishing to limit the invention to any theory or mechanism, it is believed that treatment with TGF-β1, c-ABC, and LOXL2 enhances the functional properties of the neo-cartilage to be comparable to the native articular cartilage from which the cells were sourced.

[0171] Chondrocyte isolation: Fetal ovine articular chondrocytes (foAC) were harvested from the patellofemoral surface of 120-day-pregnant Dorper-cross sheep (UC Davis School of Veterinary Medicine) obtained as medical waste. Cartilage from the entire surface of both the condyle and trochlear groove was isolated from approximately 1 mm 3After being chopped into pieces, the tissue was washed and centrifuged (500G, 5 min) three times with Dulbecco's modified Eagle's medium containing 4.5 g / L glucose and GlutaMAX (DMEM; Gibco) and 2% (v / v) penicillin / streptomycin / fungizone (PSF; Lonza). The tissue was digested in 0.2% (w / v) collagenase type II (Worthington) in DMEM containing 3% (v / v) fetal bovine serum (FBS; Atlanta Biologicals) for 18 hours at 37°C with gentle rocking. After digestion, the resulting cell solution was filtered through a 70 μm cell strainer. For studies 1-3, foACs were washed for 3 min with ACK buffer (154.4 mM ammonium chloride (Sigma), 10 mM potassium bicarbonate (Fisher Scientific), 50 mM EDTA tetrasodium salt (Acros Organics)) in ultrapure water as previously described. These primary (P0) foACs were then frozen in DMEM with 20% (v / v) DMSO (Sigma) and 10% (v / v) FBS.

[0172] Chondrocyte proliferation and redifferentiation: Previously frozen P0 foAC were cultured at 1.5 × 10 4 cells / cm 2Cells were seeded at 37 °C and grown in chemically defined chondrogenic medium (CHG medium) (DMEM containing 1% PSF, 1% ITS + premix (BD Biosciences), 1% non-essential amino acids (Gibco), 100 nM dexamethasone (Sigma), 50 mg / mL ascorbate-2-phosphate (Sigma), 40 g / mL L-proline (Sigma), and 100 mg / mL sodium pyruvate (Sigma)) with 2% FBS and chondrogenically adjusted TFP supplement (1 ng / mL TGF-β1, 5 ng / mL bFGF, 10 ng / mL PDGF; all from PeproTech). Medium was changed every 2-3 days. At confluence, cells were lifted with 0.5% trypsin-EDTA (Gibco) for 5 min, and then the cell layer was digested with DMEM containing 0.2% collagenase type II and 2% FBS at 37° C. for approximately 1 h, triturating every 20 min. The resulting cell solution was filtered through a 70 μm cell strainer and reseeded in a T-225 flask to achieve passage 3 (P3). P3 foAC underwent aggregate redifferentiation (P3R) as previously described. Briefly, 750,000 cells / mL of CHG medium containing TGB supplement (10 ng / mL TGF-β1, 100 ng / mL GDF-5, 100 ng / mL BMP-2; all from PeproTech) were cultured in 100 mm × 20 mm Petri dishes coated with 1% (w / v) molecular biology grade agarose (Thermo Fisher Scientific) made in phosphate-buffered saline (PBS; Sigma) to create a non-adherent environment. Aggregate cultures were maintained on an orbital shaker at 60 rpm for the first 3 days and left stationary for the remainder of the 14-day redifferentiation period. Medium was changed every 2-3 days. At the end of the culture period, aggregates were digested with 0.5% trypsin-EDTA for 20 min, followed by 0.2% collagenase in DMEM with 2% FBS for approximately 2 h at 37°C, disaggregating every 20 min. After dissociation of the aggregates, the cells were filtered through a 70 μm cell strainer and counted.

[0173] Visualization of chondrocyte actin: In phase 2, to visualize the effect of cytochalasin D treatment on cytoskeleton-mediated chondrogenic redifferentiation, F-actin staining was performed on untreated P0 foAC, and both cytochalasin D-treated and untreated P3 and P3R foAC. Approximately 8 × 10 3 cells / cm 2 were allowed to attach to glass slides for 1 h in the presence of 2% FBS. Non-adherent cells were washed away with two changes of PBS before the adherent cells were fixed in 3.9% formaldehyde in PBS for 10 min. After two more washes with PBS, the fixed cells were permeabilized with 0.1% Triton-X 100 (Sigma) in PBS for 5 min. After two washes with PBS, the cells were stained with CF594-conjugated phalloidin (biotin; 1:200 dilution in PBS) for 30 min. Excess stain was washed away with two changes of PBS and the cells were counterstained with DAPI-containing Vectashield (Vector Laboratories), coverslipped, and visualized using the Texas Red fluorescent channel.

[0174] Seeding and culture of neo-cartilage constructs: P0, P3, or P3R foAC were allowed to self-assemble into engineered neo-cartilage constructs in non-adherent agarose wells. Sterile stainless steel molds consisting of 5 mm diameter cylindrical posts were inserted into 48-well plates to create a single agarose well per plate well, with each well containing 1 mL of molten 2% (w / v) agarose. The molds were removed after the agarose was allowed to solidify at room temperature. The agarose wells were filled with CHG medium, which was changed twice over a period of 5 days, to ensure saturation of the agarose prior to seeding. For each phase, cells were seeded into 5 mm agarose wells in 100 μL CHG medium / well. In phase 1, P0 or P3R foAC were each seeded at five densities (2, 3, 4, 5, and 6 million cells per construct). In phase 2, P3 and P3R foAC were seeded at 2 million cells per construct. In phase 3, 2 million P3R foAC were seeded. All constructs were released on day 6 and placed into larger wells that were coated with agarose to prevent construct adhesion. Media was changed daily before release and every other day after the 6-week culture period. In phase 1, no chemical treatments were applied during neocartilage culture. In phase 2, cytochalasin D (Enzo Life Sciences; 2 μM at seeding and for the first 48 hours) and hyaluronidase (200 units / mL at seeding) were applied in a full factorial configuration. In phase 3, cytochalasin D was administered as in the previous phase, and TCL treatment consisting of TGF-β1 (10 ng / mL throughout the entire culture period), chondroitinase ABC (c-ABC, Sigma; 2 units / mL for 4 h on day 7), and a LOX cocktail consisting of lysyl oxidase-like 2 (LOXL2, Signal Chem; 0.15 μg / mL), copper sulfate (Sigma; 1.6 μg / mL), and hydroxylysine (Sigma; 0.146 μg / mL) was also administered on days 7–21. For reference, P0 foACs not treated with ACK buffer during isolation (P0 control) were seeded at 4.5 million cells per construct and received no other chemical treatments during neocartilage culture. All neocartilage evaluations were performed at the end of the culture period.

[0175] Gross Morphological Analysis of Neocartilage: Diameter and thickness of the neocartilage constructs were measured from photographs using ImageJ (National Institutes of Health). Wet weights were obtained by weighing the entire construct, and samples were then sectioned for histological, biochemical, and mechanical analysis.

[0176] Histological and immunohistochemical evaluation of neocartilage: Formalin-fixed samples were embedded in paraffin and sectioned into 5 μm sections along the short axis to expose the full thickness of the construct. In all studies, sections were stained with H&E to show morphology, Safranin O / Fast Green to show glycosaminoglycan (GAG) deposition, and Picrosirius Red to visualize collagen. Von Kossa and Alizarin Red staining were also performed to observe mineralization. Immunohistochemistry (IHC) was performed to stain for collagen I (Abcam ab34710, dilution 1:250), collagen II (Abcam ab34712, dilution 1:4000). In phase 1, IHC was also performed to stain for collagen VI (Abcam ab6588, dilution 1:250) and collagen X (Abcam ab49945, dilution 1:200).

[0177] Neocartilage biochemical evaluation: Biochemical samples were weighed to determine wet weight, lyophilized, and weighed again to determine dry weight. Dried samples were digested for 18 hours at 65°C in 125 μg / mL papain (Sigma-Aldrich), 5 mM N-acetyl-L-cysteine, 5 mM EDTA, 100 mM phosphate buffer. Glycosaminoglycan (GAG) content was measured by Blyscan assay kit (Biocolor). Collagen content was measured by a modified colorimetric chloramine-T hydroxyproline assay using hydrochloric acid. A standard curve was generated using Sircol collagen standards (Bicolor). DNA content was measured using PicoGreen dsDNA reagent (Invitrogen). Neocartilage collagen content and GAG content were normalized to wet weight, dry weight, and DNA content. Pyridinoline crosslinks were quantified by high performance liquid chromatography (HPLC) using pyridinoline standards (Quidel) as previously described. Pyridinoline content was normalized to wet weight and collagen content.

[0178] Mechanical evaluation of neocartilage: Creep indentation compression tests were performed on punches (3 mm diameter) from each construct by applying a flat porous indenter tip (0.8 mm diameter) with loads ranging from 0.45 to 2 g to achieve strains of 10 to 15%. Aggregate and shear moduli were obtained from experimental data using semi-analytical, semi-numerical, linear biphasic models, and finite element analysis. Tensile tests were performed according to ASTM standards (ASTM D3039). Constructs were punched into dog-bone shaped specimens with a gauge length of 1.92 mm, and paper tabs were attached to the tissue outside the gauge length. The paper tabs were gripped in a TestResources machine (TestResources Inc.) and pulled at 1% of the gauge length per second until the sample broke. Stress-strain curves were generated from the experimental data and the sample cross-sectional areas measured by ImageJ analysis. A least-squares fit of the linear region of the curve was used to obtain the tensile modulus, and the maximum stress gave the ultimate tensile strength (UTS).

[0179] Functionality index assessment: A modified functionality index (FI; Equation 1) was used to quantitatively evaluate engineered neocartilage in all phases against native fetal and juvenile bovine articular cartilage to select culture conditions to proceed in each phase. All factors were weighted equally based on the structure-function relationship within the cartilage, the importance of both compressive and tensile properties during joint loading, the importance of biochemical properties for tissue integration, and the contribution of crosslinks to mechanical integrity. In the functionality index, G stands for GAG / WW (%), C stands for total collagen / WW (%), P stands for pyridinoline / collagen (nmol / mg), E stands for total collagen / WW (%), F ... C represents the (compressive) aggregate modulus, and E T represents the tensile modulus. The subscripts nat and eng represent native and engineered tissue, respectively. Constructs with inconsistent thickness and abnormal morphology, e.g., tears, ruptures, or bulbous regions, were considered unsuitable and excluded from the functionality index evaluation.

[0180] Formula 1:

number

[0181] Statistical analysis: In phase 1, quantitative neocartilage properties and functional indices of different seeding densities across the two passage conditions were analyzed using two-way analysis of variance (ANOVA) in Prism 7 (GraphPad Software) followed by Tukey's post-hoc test. In phase 2, one-way ANOVA followed by Tukey's post-hoc test was performed to analyze quantitative neocartilage properties and functional indices between different treatment groups. In phase 3, Student's t-test was performed to analyze quantitative properties and functional indices between treatment groups. A sample size of n=6 per group was used. All data are presented as mean±standard deviation. Significance was determined by P<0.05 and is indicated in figures showing quantitative results by marking statistically different groups with different symbols.

[0182] Phase 1: Neo-cartilage constructs showed differences in morphology based on passage and cell density (see FIG. 6). Relative to P0 neo-cartilage, the diameter, thickness, and wet weight of the constructs increased with increasing cell seeding density. The diameters of P0 constructs seeded with 2, 3, 4, 5, and 6 million cells and P3R constructs seeded with the same cell densities were 5.3±0.2 (FIG. 6E), 6.2±0.2 (FIG. 6D), 6.9±0.2 (FIG. 6C), 7.1±0.3 (FIG. 6C), 7.2±0.1 (FIG. 6C), 8.2±0.2 (FIG. 6A), 8.2±0.1 (FIG. 6A, FIG. 6B), 7.8±0.3 (FIG. 6B), 7.2±0.1 (FIG. 6C), and 7.0±0.2 (FIG. 6C) mm, respectively. The thickness of P0 constructs seeded with 2, 3, 4, 5, and 6 million cells, and the diameter of P3R constructs seeded at the same cell densities were 0.5 ± 0.0 (Figure 6F), 0.5 ± 0.0 (Figure 6F), 0.7 ± 0.1 (Figure 6E), 0.7 ± 0.2 (Figure 6D, Figure 6E), 0.9 ± 0.1 (Figure 6B, Figure 6C), 0.9 ± 0.0 (Figure 6B, Figure 6C, Figure 6D), 1.0 ± 0.0 (Figure 6B), 1.2 ± 0.1 (Figure 6A), 0.7 ± 0.0 (Figure 6C, Figure 6D, Figure 6E), and 0.8 ± 0.1 (Figure 6B, Figure 6C, Figure 6D, Figure 6E) mm, respectively. The wet weights of P0 constructs seeded at 2, 3, 4, 5, and 6 million cells, and the diameters of P3R constructs seeded at the same cell densities were 12.8 ± 0.5 (Figure 6G), 19.0 ± 0.6 (Figure 6F), 30.2 ± 3.5 (Figure 6E), 35.2 ± 5.2 (Figure 6D), 39.5 ± 1.9 (Figure 6D), 49.5 ± 1.9 (Figure 6C), 53.6 ± 1.4 (Figure 6B, Figure 6C), 58.2 ± 1.3 (Figure 6A, Figure 6B), 59.3 ± 3.6 (Figure 6A), and 58.4 ± 4.0 (Figure 6A) mg, respectively. Constructs seeded at densities of 2, 3, and 4 million cells appeared uniform and disc-shaped and maintained a consistent thickness within each construct. Although of consistent thickness, the 2 and 3 million cell constructs were curved, while the 4 million cell constructs were flat. Constructs seeded at 5 and 6 million cells showed inconsistent thickness and irregular morphology, including folded and broken edges.In P3R neocartilage, in general, as seeding density increased, construct diameter decreased while thickness and wet weight increased. Constructs seeded with 4 million cells showed small, distinct pockets of less cellular diffuse matrix. At seeding densities of 5 and 6 million cells, these areas ruptured, causing the construct to form two distinct layers, with only one layer remaining completely intact. The thicknesses reported for these constructs were measured from the intact layer.

[0183] Histologically, differences in cell morphology and the intensity of GAG, collagen, and collagen II staining as a function of passage and neocartilage seeding density were observed (see FIG. 6). H&E staining revealed larger chondrocytes in both P0 and P3R constructs than those present in the native tissue. In addition, the lacunae surrounding the cells in the P3R construct were larger than those in the P0 neocartilage. Safranin O staining for sulfated GAGs showed stronger staining in both P0 and P3R constructs compared to the native tissue. Safranin O stained less strongly in the outer region of the P0 construct compared to the central region. This outer region was greatly reduced in the P3R construct. GAG staining appeared strongest at a seeding density of 4 million cells in P0 neocartilage. In P3R neocartilage, GAG staining was strongest at a density of 2 million cells and decreased with increasing seeding density. Picrosirius red staining for collagen was less intense in both P0 and P3R neotissue compared to native tissue. The outer regions of the P0 and P3R constructs stained more intensely than the inner regions, and these regions were fainter in the P3R neocartilage. Picrosirius red staining was strongest at a seeding density of 4 million cells in P0 neocartilage and 2 million cells in P3R neocartilage. Collagen I staining was minimal across all groups. In P0 neocartilage, collagen II staining peaked at a seeding density of 4 million cells. In P3R neocartilage, collagen II staining was strongest at a seeding density of 2 million cells and decreased with increasing seeding density. Additional staining for collagen VI, collagen X, alizarin red, and Von Kossa is shown in FIG. 7. Both P0 and P3R neocartilage stained positively for collagen VI, with P3R neocartilage staining darkest. Also, P0 and P3R neocartilage stained faintly for collagen X within the lacunae, but not for the ECM surrounding the neocartilage. Neocartilage at all passages and seeding densities did not stain with Alizarin Red or Von Kossa. Calculations of biochemical content, mechanical properties, and functionality indices are listed in Table 1 of FIG. 13 and shown in FIG. 6.Functionality index identified optimal P0 (P0 Opt) and P3R (P3R Opt) seeding densities as 4 million and 2 million cells / construct, respectively. Based on superior functionality index, the P3R group seeded at 2 million cells / construct was advanced to Phase 2.

[0184] For reference, neocartilage grown from P0 foAC that was not treated with ACK buffer during isolation was also mechanically tested. These constructs were seeded at a density of 4.5 million cells / construct based on the method in the previous study with P0 foAC. Aggregate modulus, shear modulus, and permeability were 97.7 ± 20.4 kPa, 43.1 ± 12.1 kPa, and 45.1 ± 15.7 × 10, respectively. 15 m 4 The tensile modulus and UTS were 0.8±0.2 MPa and 0.2±0.1 MPa, respectively.

[0185] Phase 2: In the first study of this phase, cytochalasin D and hyaluronidase were examined to determine whether chemical treatments could redifferentiate passaged chondrocytes without aggregate redifferentiation. P3 neocartilage constructs showed significant morphological differences from P3R neocartilage. In P3 neocartilage (see FIG. 12), cytochalasin D (Cyto D) treatment resulted in a simply flat and uniform construct. No treatment (untreated), hyaluronidase treatment (Hya), or dual treatment (Hya+Cyto D) resulted in round neocartilage with diffuse void spaces in the center of the construct. The diameters of the untreated, cytochalasin D-treated, and dual-treated constructs were 2.8±0.2, 3.7±0.2, 2.6±0.1, and 3.4±0.2 mm, respectively. The diameter of the neocartilage treated with cytochalasin D was significantly greater than that of the untreated neocartilage, the hyaluronidase-treated neocartilage, and the double-treated neocartilage. The diameter of the double-treated neocartilage was also significantly greater than that of the untreated neocartilage and the hyaluronidase-treated neocartilage. The thickness of the neocartilage obtained from the untreated, hyaluronidase-treated, or double-treated was 2.1±0.2, 0.4±0.1, 2.0±0.2, and 1.5±0.7 mm, respectively. The neocartilage treated with cytochalasin D was significantly thinner than the neocartilage of the other groups. The wet weights of the untreated, cytochalasin D-treated, and double-treated neocartilage were 7.1±0.6, 8.4±1.0, 6.7±0.4, and 10.1±1.3 mg, respectively. The wet weight of the double-treated group was significantly greater than the other groups. Also, the wet weight of the cytochalasin-treated group was significantly greater than that of the untreated and hyaluronidase-treated groups. Histologically, there were void areas in the untreated, hyaluronidase-treated, and double-treated groups (see FIG. 12). All treatments except hyaluronidase produced staining darker than native fetal bovine articular cartilage. Total collagen staining for all groups was less intense than that for the native control. Cytochalasin D treatment produced the most intense GAG ​​and total collagen staining. All constructs stained for collagen I comparable to native fetal ovine meniscus, with particularly intense staining in the untreated, hyaluronidase-treated, and double-treated neocartilage around the medial diffuse area.All constructs stained minimally for collagen II. P3 biochemical and mechanical data are shown in Table 2 of Figure 14. Cytochalasin D and hyaluronidase treatment without aggregate redifferentiation failed to reduce collagen I production or increase collagen II production in P3 neocartilage.

[0186] In the second study of this phase, hyaluronidase treatment of P3R Opt neocartilage was carried forward from phase 1, introducing aggregate regeneration in conjunction with cytochalasin D. For P3R neocartilage (see FIG. 8), P3R Opt, cytochalasin D treatment (Cyto D), and dual treatment (Cyto D+Hya) resulted in constructs of uniform thickness. Hyaluronidase treatment (Hya) resulted in the formation of a diffuse void area in the center of the construct. All constructs, except the dual treatment group, were slightly bowl-shaped. The diameters of the untreated, cytochalasin D-treated, and dual treatment constructs were 8.2±0.2, 6.5±0.2, 5.9±0.0, and 5.7±0.3 mm, respectively. The construct diameters of the untreated group were significantly larger than those of the other treatment groups. The construct diameter of the cytochalasin D-treated group was significantly larger than that of the hyaluronidase and dual-treated groups. The thickness of the neocartilage obtained from untreated, cytochalasin D-treated, hyaluronidase-treated, and dual-treated was 0.9±0.0, 0.7±0.1, 0.8±0.2, and 0.4±0.1 mm, respectively. The thickness of the dual-treated neocartilage was significantly smaller than that of the other treatment groups. The wet weights of untreated, cytochalasin D-treated, and dual-treated neocartilage were 50.1±3.2, 28.1±2.6, 23.9±1.4, and 11.8±3.9 mg, respectively. Histologically, diffuse void areas were present only in the hyaluronidase-treated group (see FIG. 8). GAG, total collagen, and collagen II staining were most intense in the cytochalasin D-treated neocartilage. Biochemical and mechanical data, as well as functionality indices, are shown in Figures 8 and 15 (Table 3). Based on the superior functionality indices, cytochalasin D treatment was selected to proceed to Phase 3.

[0187] Fluorescent staining of F-actin in chondrocytes showed striking differences between cell passages and treatments (see Figure 9). In P0 chondrocytes, actin arrangement was cortical and appeared as a ring around each cell. Untreated P3 chondrocytes were much larger in size and showed a fibroblast-like intracellular fibrillar actin arrangement. Cytochalasin D treatment of P3 chondrocytes induced a rounded cell shape and while actin was still present throughout the cell, it was mostly localized to the periphery. Untreated P3R chondrocytes showed a cortical actin arrangement with some small fibrillar areas. Cytochalasin D treatment of P3R chondrocytes localized more actin to the cortex than in untreated P3R cells.

[0188] Phase 3: P3R Opt was selected as the optimal group from phase 1, and cytochalasin D treatment of P3R Opt neocartilage was selected from phase 2, and phase 3 investigated the effect of adding TCL treatment on cytochalasin D treated P3R Opt neocartilage. Neocartilage treated with both cytochalasin D and TCL appeared similar in shape to and thicker than neocartilage treated with cytochalasin D. The diameters of cytochalasin D treated neocartilage and dual cytochalasin D and TCL treated neocartilage were 6.5±0.4 and 6.4±0.3 mm, respectively. The thickness of dual treated neocartilage was significantly greater than that of cytochalasin D treated neocartilage (1.1±0.1 and 0.8±0.2 mm, respectively). The wet weights of cytochalasin D treated neocartilage and dual treated neocartilage were 87.1±3.6 and 88.8±1.3 mg, respectively. Histologically, the neocartilage of both groups appeared homogenous (see FIG. 10). Both groups stained more strongly for GAGs and less strongly for total collagen than native fetal articular cartilage. The dual treatment group stained more strongly for GAGs, total collagen, and collagen II. Neither group stained for collagen I. Biochemical and mechanical data, as well as functional index, are shown in Figures 10 and 16 (Table 4). Functional index showed that neocartilage treated with both cytochalasin D and TCL was superior to neocartilage treated with cytochalasin D alone.

[0189] Example 4 describes how, by mimicking key salient aspects of in vitro tissue formation using purified and then highly passaged cells, neocartilage was obtained with mechanical properties equivalent to the native articular cartilage from which the cells were sourced. The progressive development of neocartilage functionality through phases 1-3 is shown in FIG. 11 and demonstrates a large increase in mechanical properties. In detail, neocartilage aggregate modulus, shear modulus, and tensile modulus were found to increase 9.6-fold, 7.2-fold, and 3.8-fold over P0 controls, while tensile strength increased 9.0-fold. The neocartilage resulting from these successive studies achieved an FI of 1.42 when compared to native fetal cartilage and an FI of 1.03 when compared to native juvenile cartilage. This indicates that the engineered neocartilage exceeded native tissue values ​​for the parameters measured by the functionality index, indicating that it is possible to achieve adult-level properties.

[0190] In phase 1, neocartilage derived from P3R cells was able to achieve P0 neocartilage properties. The functional index of P3R neocartilage seeded at optimal density was comparable to that of P0 neocartilage seeded at optimal density. For fetal ovine articular cartilage, P0 Opt achieved an FI of 0.77 and P3R Opt achieved an FI of 0.78. The use of multiple passaged cells to engineer functionally robust tissue has a major translational impact as it shows that fewer cells can be isolated to engineer superior neocartilage. Hence, P3R Opt neocartilage was advanced to subsequent phases. In phase 2, it was shown that only cytochalasin D treatment was required to produce superior neocartilage from passaged / redifferentiated cells. For example, cytochalasin D treatment of P3R Opt neocartilage resulted in a 0.9-fold increase in compressive stiffness, a 1.0-fold increase in tensile stiffness, and a 2.7-fold increase in tensile strength, resulting in an FI of 1.1 versus native fetal cartilage and 0.83 versus native juvenile cartilage. Thus, cytochalasin D treatment of P3R Opt neocartilage was pursued. In phase 3, the addition of TCL treatment was shown to promote cross-linking-based maturation to enhance the functional properties of the neocartilage achieving an FI of 1.42 versus native fetal cartilage and 1.03 versus native juvenile cartilage. The aggregate modulus exceeded that of native fetal cartilage, and the tensile modulus was within the range of native levels. This study represents an important step toward achieving biomimetic articular cartilage and using multiple passage cells to do so.

[0191] In phase 1, P3R Opt neocartilage achieved comparable FI to P0 Opt neocartilage. In P0 neocartilage, the functional properties of the construct increased with increasing seeding density until a plateau was reached. However, in P3R neocartilage, the functional properties decreased with increasing seeding density. This is in contrast to conventional tissue engineering strategies that assume that primary cells are more synthetically capable than passaged cells and that higher cell numbers are required to generate superior neotissue. In this study, chondrocytes were expanded over 4,000-fold and seeded at a lower density than required for primary cells to achieve neocartilage with a larger diameter and comparable FI. The aggregate modulus, GAG / DNA production, and collagen / DNA production of P3R Opt neocartilage were 0.3-, 2.2-, and 2.6-fold greater than those of P0 Opt neocartilage (see FIG. 6). Although the collagen content in P3R Opt neocartilage was comparable to that of P0 Opt neocartilage, the tensile stiffness and strength of P3R Opt neocartilage were significantly reduced. Given the importance of collagen crosslinks to the tensile properties of cartilage, the reduced pyridinoline content in P3R Opt neocartilage was likely the reason for this, as addressed by TCL treatment in phase 3. By using chondrogenically tuned proliferation and aggregate redifferentiation methods and optimizing self-assembling culture conditions, it was possible to engineer robust neocartilage from multi-passaged cells. 8,000-fold fewer primary cells were required to achieve this than engineering neocartilage from non-passaged cells.

[0192] It was unexpected that by examining multiple seeding densities across passaging conditions, passaged chondrocytes could be recalibrated to exhibit more immature behavior. At seeding densities of 2, 3, and 4 million cells, P3R chondrocytes were more synthetic than P0 chondrocytes. Example 4 mimicked the proliferation, condensation, differentiation, and tissue formation that occurs developmentally through in vitro steps such as monolayer growth, aggregate redifferentiation, and self-assembly. Evidence suggests that by doing so, P3R chondrocytes were recalibrated to a more immature state, allowing for increased production of matrix molecules. For example, the matrix secreted by P3R chondrocytes better reflected the composition of articular cartilage ECM at early stages. As native cartilage matures, collagen VI staining present throughout the ECM is localized to the pericellular matrix and collagen II staining increases. Also, pyridinoline content in native cartilage increases significantly over time as cartilage matures. In this study, P3R neocartilage showed weaker collagen II staining, stronger collagen VI staining, and lower levels of pyridinoline compared to P0 neocartilage (see Figures 6 and 7). These data support the assertion that P3R chondrocytes are in a more immature state than P0 chondrocytes.

[0193] A culture technique called polymer crowding has been used to enhance the generation and maturation of cartilage matrix by chondrocytes in monolayers, but shows negative effects in 3D cultures. When Ficoll 70 and Ficoll 40 were used with chondrocytes in monolayers, collagen II expression, as well as GAG and total collagen production, were increased. However, in the 3D pellet culture model, polymer crowding treatment led to degradation of the cartilage matrix as early as day 2 of culture. In addition, the proinflammatory cytokine IL-6 was detected in the medium of high-density cultures but not in low-density cultures. This study showed that P3R chondrocytes have a high synthetic potential. Matrix deposition in self-assembling neocartilage is known to begin as early as day 1 after cell seeding.

[0194] Passaged chondrocytes show a strong chondrogenic phenotype in self-assembled neocartilage. With redifferentiated chondrocytes and the newly proposed mechanisms of self-induced macromolecular crowding and inflammatory cytokine-regulated matrix production, confirmation of the phenotype of chondrocytes in culture is necessary. In osteoarthritis, chondrocytes show proliferation, increased synthesis of matrix molecules including collagen X, hypertrophy, and mineralization. To confirm the chondrogenic phenotype of P3R neocartilage, P3R neocartilage and P0 neocartilage were stained for collagen VI, collagen X, alizarin red, and von Kossa (see Figure 7). P3R neocartilage stained more intensely for collagen VI than P0 neocartilage. All groups were faintly stained in the tear cavity for collagen X, indicating that its presence was not caused by in vitro manipulations such as cartilage-conditioned proliferation and aggregate redifferentiation performed in this study. No groups were stained with alizarin red or von Kossa, indicating that there was no mineralization. The presence of collagen X, in addition to the large lacunae observed in P3R neocartilage, may indicate a degenerative tissue or hypertrophic chondrocyte phenotype, while calcification was absent. In addition, the size of the lacunae is reduced in dense P3R neocartilage. As collagen VI, collagen X, and large lacunae are indicative of immature cartilage, these data further support that a recalibration of the passaged chondrocytes to an immature phenotype has been achieved.

[0195] In phase 2, cytochalasin D treatment of passaged / redifferentiated cells further enhanced the chondrogenic phenotype. Cytochalasin D treatment of P3R Opt neocartilage (progressed from phase 1) increased aggregate modulus by 0.9-fold over the untreated group (see FIG. 8) and to the level of native articular cartilage of adult sheep. Tensile stiffness and strength also increased by 1.0-fold and 2.7-fold, respectively, potentially due to a small concomitant increase in collagen and pyridinoline content. This treatment resulted in neocartilage with an FI of 1.1 over native fetal cartilage and 0.83 over native juvenile cartilage. The success of this treatment on passaged / redifferentiated cells motivated the study of the effect on passaged non-redifferentiated (P3) chondrocytes. Cytochalasin D treatment of P3 neocartilage resulted in simply flat, uniform constructs of all treatments (see FIG. 12). However, its action alone was not sufficient to affect redifferentiation to a degree sufficient to reveal changes in the functional properties of the constructs, as indicated by the strong collagen I staining, as well as the lower biochemical content and mechanical properties (see FIG. 14 (Table 2)). Functionality indexes were not calculated for P3 neocartilage of either treatment, as the constructs were not testable under tension and were not morphologically acceptable. Visualization of actin in P3R and P3 chondrocytes confirmed a similar mode of action of cytochalasin D as observed with P0 cells (see FIG. 9). Cytochalasin D treatment significantly improved the cortical organization of F-actin in P3 and P3R chondrocytes.

[0196] Phase 3 mimicked the progression of tissue formation by enhancing neocartilage matrix deposition and crosslinking to achieve native levels of tensile properties. In phase 1, pyridinoline / WW was significantly reduced in P3R neocartilage compared to P0 neocartilage. These levels remained consistently low in phase 2. This prevents the neocartilage from achieving improved tensile properties, while the slow development of pyridinoline crosslinks compared to other matrix components mimics native cartilage maturation. Phase 3 used TCL treatment, which has been shown to increase collagen content and crosslinks within the collagen network. This treatment indeed resulted in a 0.9-fold increase in collagen / WW and a 2.9-fold increase in pyridinoline / WW, as well as a 1.7-fold increase in tensile stiffness and a 3.5-fold increase in tensile strength, without changing the compressive stiffness. These tensile properties are within the range reported for juvenile ovine articular cartilage. Additionally, TCL-treated neocartilage achieved an FI of 1.42 versus native fetal cartilage and 1.03 versus native juvenile cartilage, indicating that the properties of the neocartilage engineered in this study are now approaching adult levels. Mimicking key steps in native cartilage formation and following the developmentally suggested sequence of matrix development and maturation enabled purified, passaged / redifferentiated cellular neocartilage to achieve tensile properties within the range of native cartilage.

[0197] By using developmentally suggested chemical stimuli, mimicking key aspects of native chondrogenesis, this study was able to engineer neocartilage from cells expanded over 4,000-fold with functional properties approaching native adult cartilage. Treatment with ACK lysis buffer, cytochalasin D, and TCL, in addition to chondrogenically tuned proliferation, redifferentiation of aggregates, and optimized self-assembly of neocartilage, resulted in mature neocartilage with the highest functional index reported by our group. In addition, it was shown that passaged cells could be recalibrated to a more synthetic state. A mechanism based on self-induced macromolecular crowding and cytokine-regulated feedback inhibition of cartilage matrix synthesis in high-density 3D cultures provides a plausible explanation for seeding density-dependent matrix synthesis. Finally, an updated functional index was provided that accounts for the importance of tissue bridging. This study is well on its way to establishing a protocol for generating native-like engineered neocartilage from 8,000-fold fewer primary cells than previous methods.

[0198] Example 5 - Shear Example 5 describes a method to select cells based on membrane properties using shear. Example 5 shows a protocol for purifying articular chondrocytes using shear.

[0199] Cell isolation: Fetal ovine AC are isolated from the femoral condyles and trochlear grooves of the knees of 120-125 day pregnant Dorper-cross sheep (UC Davis School of Veterinary Medicine). Minced cartilage tissue is washed with PBS and digested with 500 units / mL collagenase type 2 (Worthington Biochemical, Lakewood, NJ) in chondrogenic medium + 3% (v / v) FBS (Atlanta Biologicals, Lawrenceville, GA) for 18 hours at 37°C / 10% CO2. Cells are then strained through a 70 μm filter, washed with wash medium, and counted.

[0200] Protocol for introducing shear to purify chondrocytes: (1) Place approximately 50 mL of cell solution into a Petri dish. (2) Submerge the paddle rotor into the Petri dish and spin at 20 rpm for 3 minutes. (3) Remove the paddle rotor and wash the processed cell solution twice with washing medium to count the remaining cells.

[0201] Example 6 - Impact / Compression Example 6 describes a method to select cells based on membrane properties using impact / compression. Example 6 shows a protocol for purifying articular chondrocytes using compression / impact.

[0202] Cell isolation: To obtain costal chondrocytes, 1–2 mm cartilage from juvenile bovine stifle joints was cut into pieces. 3 The cells are minced into pieces and digested in 0.2% type II collagenase (Worthington) in Dulbecco's modified Eagle's medium (DMEM) (Gibco) with 1% penicillin / streptomycin / fungizone (PSF) (BD Biosciences) and 3% fetal bovine serum (Atlanta Biologicals) for 18 hours at 37° C. After digestion, the chondrocytes are filtered through a 70 μm cell strainer, resuspended in blank DMEM, and counted.

[0203] Protocol for introducing compression / impact to purify chondrocytes: (1) Place approximately 20 mL of cell solution into a conical tube. (2) Centrifuge the cell solution at 300 g for 5 min to form a pellet. (3) Insert the associated mesh conical pestle. Ensure that the mesh size is less than 15 μm. Gently compress the cell pellet with the mesh pestle once every 30 seconds for 3 min. (4) Remove the pestle and wash the processed cell solution twice with washing medium to count the remaining cells.

[0204] C. Purification based on membrane surface area properties Example 7 - Hypotonic Solution Example 7 describes a method for selecting cells based on stiffness using a hypotonic solution. Without wishing to limit the invention to any particular theory or mechanism, it is believed that treatment of chondrocytes with a hypotonic buffer is effective in increasing viable chondrocyte purity by reducing the population of cells with existing undesirable stiffness properties. Thus, this treatment increases the functional properties of the resulting self-assembling neocartilage by generating a population of cells enriched for viable chondrocytes without undesirable stiffness properties.

[0205] Example 7 demonstrates that native cartilage compressibility is achieved in engineered neocartilage.The present invention is not limited to the methods or compositions described herein.

[0206] In Example 7, chondrocytes were isolated from the stifle joint of fetal sheep, a highly clinically translatable cell source. First, treatment of primary (P0) fetal chondrocytes with ammonium chloride-potassium lysis buffer (ACK buffer) was examined to determine the effect on chondrocyte purity within the cell isolate and on the functional properties of the resulting self-assembling neocartilage. Chondrocyte purity was assessed by cell counting. The functional properties of the neocartilage were evaluated by a standard set of assays including compressive creep indentation, uniaxial tensile testing, GAG, collagen, and DNA assays, as well as histology and IHC. Second, the seeding density of P0 and passage-redifferentiated (P3R) fetal chondrocytes during the self-assembly method was examined. Cells were seeded at 2, 3, 4, 5, and 6 million cells per 5 mm construct, and the functional properties of the resulting neocartilage were evaluated using the same set of assays. Finally, cytochalasin D and hyaluronidase were used in a full factorial design at the start of the self-assembly process to investigate their potential to further enhance the functional properties of the resulting neocartilage, which was evaluated by a standard battery of assays.

[0207] Results: ACK buffer treatment of freshly isolated P0 fetal chondrocytes reduced red blood cell contamination in cell isolation by 60%. ACK treatment significantly increased neocartilage 1) aggregate modulus by 1.8-fold, 2) shear modulus by 1.3-fold, and 3) tensile modulus by 0.8-fold. Continuing with anterior ACK treatment of chondrocytes during isolation, the seeding density of P0 chondrocytes was optimized to 4 million cells / construct, additionally increasing neocartilage aggregate modulus by 0.6-fold and shear modulus by 0.8-fold. After passaging and redifferentiation (P3R) of these cells, the seeding density was optimized to 2 million cells / construct, further increasing aggregate modulus by 0.3-fold and shear modulus by 0.3-fold. The use of cytochalasin D during self-assembly of ACK-treated P3R chondrocytes seeded at 2 million cells / construct significantly increased the neocartilage aggregate modulus to 400 kPa, 9.6-fold over P0 controls.

[0208] As discussed above, the present invention features methods for engineering cartilage with compressibility generally similar to native cartilage, comprising purifying isolated chondrocytes (e.g., via hypotonic lysis buffers), optimizing neo-cartilage seeding density, redifferentiating passaged chondrocytes via novel aggregate culture methods such that primary cell neo-cartilage properties are preserved, and / or enhancing chondrocyte activity via cytoskeleton modifying agents.

[0209] Example 8 - Shear Example 8 describes a method to select cells based on stiffness using shear. Example 8 shows a protocol for purifying articular chondrocytes using shear. Cell isolation: Fetal bovine articular chondrocytes (foAC) are isolated from the stifle joints of Dorper cross sheep at 120 days of gestation. Cartilage from the condyles and trochlear groove is isolated at approximately 1 mm 3The tissue is chopped into pieces, washed and centrifuged (500G, 5 min) three times with Dulbecco's modified Eagle's medium containing 4.5 g / L glucose and GlutaMAX (DMEM; Gibco) and 2% (v / v) penicillin / streptomycin / fungizone (PSF; Lonza). The tissue is digested in 0.2% (w / v) collagenase type II (Worthington) in DMEM containing 3% (v / v) fetal bovine serum (FBS; Atlanta Biologicals) for 18 hours at 37°C with gentle rocking. After digestion, the resulting cell solution is filtered through a 70 μm cell strainer.

[0210] Protocol for introducing shear to purify chondrocytes: (1) Take the cell solution into a sterile 10 mL syringe. (2) Attach the syringe to a microfluidic device with channels of 75 μm to 200 μm in diameter. (3) Slowly depress the syringe plunger to allow the cell solution to flow through the microfluidic device into the conical tube reservoir. (4) Depress the syringe fully to inject another 20 mL of DMEM into the microfluidic device. (5) Wash the processed cell solution twice with washing medium and count the remaining cells.

[0211] Example 9 - Impact / Compression Example 9 describes a method to select cells based on stiffness using impact / compression. Example 9 shows a protocol for purifying articular chondrocytes by applying compression / impact. Cell isolation: Juvenile bovine articular chondrocytes are harvested from the patellofemoral surface of bovine stifle joints. Articular cartilage is extracted from approximately 1 mm 3The tissue is chopped into pieces, washed three times with Dulbecco's modified Eagle's medium containing 4.5 g / L glucose and GlutaMAX (DMEM; Gibco) and 2% (v / v) penicillin / streptomycin / fungizone (PSF; BD Biosciences) and centrifuged (500 G, 5 min). The chopped tissue is digested for 18 hours at 37° C. in 0.2% (w / v) collagenase type II (Worthington) in DMEM containing 3% (v / v) fetal bovine serum (FBS; Atlanta Biological). After digestion, the resulting cell solution is filtered through a 70 μm cell strainer, centrifuged (500 G, 5 min), and resuspended in blank DMEM.

[0212] Protocol for introducing impact / compression to purify chondrocytes: (1) Place approximately 50 mL of cell solution in a Petri dish. Add 20 glass beads of 0.25-0.5 mm to the Petri dish. (2) Submerge the paddle rotor in the Petri dish and spin at 20 rpm for 3 minutes. (3) Remove the paddle rotor and pipette the cell solution into a conical tube. (4) Wash the glass beads with 50 mL DMEM and place the washed DMEM into the conical tube with the processed cell solution. (5) Wash the processed cell solution twice with wash medium and count the remaining cells.

[0213] Example 10 Example 10 describes the enhancement of translatability of purified and expanded chondrocytes to engineer native-like neocartilage.The present invention is not limited to the methods or compositions described herein.

[0214] Chondrocytes were isolated from the stifle joints of fetal sheep, as fetal cells represent a highly clinically relevant cell type for tissue engineering. First, ACK buffer treatment of primary (P0) chondrocytes reduced red blood cell contamination by 60% and increased neocartilage aggregate modulus (1.8-fold), shear modulus (1.3-fold), and tensile modulus (0.8-fold). Then, seeding density optimization of proliferated / redifferentiated (P3R) chondrocytes to 2 million cells / construct further increased aggregate modulus (1.0-fold) and shear modulus (1.1-fold). Finally, cytochalasin D treatment further increased neocartilage aggregate modulus by 9.6-fold over untreated P0 controls, to 400 kPa, equivalent to native cartilage. Remarkably, P3R cells treated with ACK buffer and cytochalasin D resulted in neocartilage with compressibility exceeding that of P0 neocartilage and similar to native cartilage. These successive studies allowed for 4000-fold fewer primary cells to be used to engineer robust neocartilage, specifically 1000 primary cells per P3R construct versus 4,000,000 primary cells per P0 construct, greatly enhancing the clinical translatability of expanded chondrocytes for tissue engineering.

[0215] Example 11 Example 11 describes the method of cell isolation and processing. The present invention is not limited to the methods and compositions described below, for example, the present invention is not limited to providing chondrocytes from rib cartilage tissue, the present invention is not limited to ACK buffer concentration, etc. As mentioned above, chondrocytes can be provided from other cartilage tissues.

[0216] A sample is isolated from human rib cartilage tissue. The rib cartilage tissue is cut to remove muscle, fat tissue, and perichondrium so that essentially only cartilage remains. The harvested rib cartilage is further cut into small pieces, then centrifuged and washed. The rib cartilage pieces are then subjected to an enzyme digestion step, for example, by incubating the rib cartilage pieces with the enzyme for a period of time with gentle rocking to obtain a single cell suspension.

[0217] The cell suspension is then subjected to a hypotonic buffer. For example, the cell suspension is subjected to a pre-warmed ACK buffer, which includes ammonium chloride, potassium bicarbonate, and tetrasodium salt of EDTA. In some embodiments, the concentration of the components of the ACK buffer includes, but is not limited to, 154.4 mM ammonium chloride, 10 mM potassium bicarbonate, and 97.3 μM tetrasodium salt of ethylenediaminetetraacetic acid (EDTA). The cell suspension is then centrifuged, the supernatant is aspirated, and the pellet is resuspended in a hypotonic buffer (ACK buffer, which includes ammonium chloride, potassium bicarbonate, and tetrasodium salt of EDTA). The cell suspension is then incubated with the hypotonic (ACK) buffer for a period of time, and then centrifuged. The supernatant is aspirated, and the cell pellet is washed and then plated, for example, for passage.

[0218] In some embodiments, the harvested rib cartilage is separated into approximately 1 mm 3 In some embodiments, the washing step includes Dulbecco's Modified Eagle's Medium containing 4.5 g / L glucose and GlutaMAX (DMEM; Gibco) and 2% (v / v) penicillin / streptomycin / fungizone (PSF; BD Biosciences). In some embodiments, the digestion step includes the use of enzymes such as, but not limited to, pronase and / or type II collagenase. In some embodiments, the cell pellet is frozen instead of plating for passaging.

[0219] Various modifications of the present invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description, and such modifications are intended to fall within the scope of the appended claims. Each reference cited in this application is incorporated herein by reference in its entirety.

[0220] While preferred embodiments of the present invention have been shown and described, it will be readily apparent to those skilled in the art that modifications may be made therein that do not exceed the scope of the appended claims. Accordingly, the scope of the present invention should be limited only by the following claims.

[0221] In some embodiments, the description of the invention set forth herein using the phrase "comprising" encompasses embodiments that may be described as "consisting of," thereby satisfying the description requirement for claiming one or more embodiments of the invention using the phrase "consisting of."

[0222] Embodiment

[0223] The following embodiments are intended to be illustrative only and not limiting in any way.

[0224] Embodiment 1A: A method of enriching a sample of chondrocytes comprising: (a) obtaining a sample of chondrocytes comprising a mixed population of non-pre-apoptotic and pre-apoptotic cells; and (b) subjecting the sample of chondrocytes derived from (a) to a treatment, which may be repeated multiple times, alone or in combination with other treatments.Embodiment 2A: A method of preparing a sample of chondrocytes comprising: (a) obtaining a sample of chondrocytes comprising a mixed population of non-pre-apoptotic and pre-apoptotic cells; and (b) subjecting the sample of chondrocytes derived from (a) to a treatment, which may be repeated multiple times, alone or in combination with other treatments.Embodiment 3A: The method of embodiment 1A or embodiment 2A, wherein the treatment comprises adding a hypotonic solution.Embodiment 4A: A method of enriching a sample of chondrocytes comprising: (a) obtaining a sample of chondrocytes comprising a mixed population of non-pre-apoptotic and pre-apoptotic cells; and (b) subjecting the sample of chondrocytes derived from (a) to a treatment with a hypotonic solution, which may be repeated multiple times, alone or in combination with other treatments. Embodiment 5A: A method of preparing a sample of chondrocytes comprising: (a) obtaining a sample of chondrocytes comprising a mixed population of non-pre-apoptotic and pre-apoptotic cells; and (b) subjecting the sample of chondrocytes derived from (a) to a treatment with a hypotonic solution, which may be repeated multiple times, alone or in combination with other treatments.Embodiment 6A: The method of any one of embodiments 3A-5A, wherein said hypotonic solution is an ammonium chloride potassium lysis buffer (ACK buffer).Embodiment 7A: A method of enriching a sample of chondrocytes comprising: (a) obtaining a sample of chondrocytes comprising a mixed population of non-pre-apoptotic and pre-apoptotic cells; and (b) subjecting the sample of chondrocytes derived from (a) to a treatment with an ammonium chloride potassium lysis buffer (ACK buffer); which may be repeated multiple times, alone or in combination with other treatments.Embodiment 8A: A method of preparing a sample of chondrocytes, comprising: (a) obtaining a sample of chondrocytes comprising a mixed population of non-pre-apoptotic and pre-apoptotic cells; and (b) subjecting the sample of chondrocytes derived from (a) to a treatment with ammonium chloride potassium lysis buffer (ACK buffer); which may be repeated multiple times, alone or in combination with other treatments. Embodiment 9A: The method of any one of embodiments 1A-8A, wherein said treatment induces cell swelling. Embodiment 10A: The method of any one of embodiments 1A-9A, wherein said sample of chondrocytes is a sample of human chondrocytes. Embodiment 11A: The method of any one of embodiments 1A-10A, wherein said sample of chondrocytes is a sample of non-articular chondrocytes. Embodiment 12A: The method of any one of embodiments 1A-11A, wherein said sample of cells is provided from a portion of a rib. Embodiment 13A: The method of embodiment 12A, wherein said rib comprises chondrocytes. Embodiment 14A: The method of embodiment 13A, wherein said chondrocytes are non-articular chondrocytes. Embodiment 15A: The method of any one of embodiments 1A-14A, wherein after (b), the sample of cells has a higher percentage of non-pre-apoptotic cells compared to the sample of cells before (b).Embodiment 16A: The method of any one of embodiments 1A-14A, wherein after (b), the sample of cells has a lower percentage of pre-apoptotic cells compared to the sample of chondrocytes before (b).Embodiment 17A: The method of any one of embodiments 1A-16A, wherein the cells generated after (b) are used for one or more of the following: direct use of cells; in vitro culture of cells including passaging in a monolayer or three-dimensional environment, including suspension culture; tissue engineering using scaffold-free systems including self-assembly or using scaffold-based systems including natural and synthetic materials; cell transplantation; tissue transplantation; and / or grafting.Embodiment 18A: The method of embodiment 1A, further comprising passaging the cells in a monolayer or three-dimensional environment after (b). Embodiment 19A: The method of embodiment 18A, further comprising generating neocartilage with said passaged cells.Embodiment 20A: The method of any one of embodiments 1A-19A, wherein the cells generated after (b), or tissue engineered / created from said cells, are subjected to a treatment comprising one or more of the following: growth factors; cytoskeletal modifying agents; hormones; toxic compounds; molecules acting upstream of a signaling cascade; varying oxygen tension; cross-linking agents; matrix-degrading enzymes, matrix molecules; and / or mechanical stimulation.

[0225] Embodiment 1B: A method for enriching a sample of human chondrocytes, comprising: (a) obtaining a sample of human chondrocytes comprising a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) subjecting the sample of chondrocytes derived from (a) to a treatment, which may be repeated multiple times, alone or in combination with other treatments.Embodiment 2B: A method for preparing a sample of human chondrocytes, comprising: (a) obtaining a sample of human chondrocytes comprising a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) subjecting the sample of chondrocytes derived from (a) to a treatment, which may be repeated multiple times, alone or in combination with other treatments.Embodiment 3B: The method of embodiment 1B or embodiment 2B, wherein said treatment comprises adding a hypotonic solution.Embodiment 4B: A method for enriching a sample of human chondrocytes, comprising: (a) obtaining a sample of human chondrocytes comprising a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) subjecting the sample of human chondrocytes derived from (a) to a treatment with a hypotonic solution, which may be repeated multiple times, alone or in combination with other treatments. Embodiment 5B: A method of preparing a sample of human chondrocytes, comprising: (a) obtaining a sample of human chondrocytes comprising a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) subjecting the sample of human chondrocytes derived from (a) to a treatment with a hypotonic solution, which may be repeated multiple times, alone or in combination with other treatments.Embodiment 6B: The method of any one of embodiments 3B-5B, wherein said hypotonic solution is an ammonium chloride potassium lysis buffer (ACK buffer).Embodiment 7B: A method of enriching a sample of human chondrocytes, comprising: (a) obtaining a sample of human chondrocytes comprising a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) subjecting the sample of human chondrocytes derived from (a) to a treatment with a hypotonic solution, which is an ammonium chloride potassium lysis buffer (ACK buffer); which may be repeated multiple times, alone or in combination with other treatments.Embodiment 8B: A method of preparing a sample of human chondrocytes, comprising: (a) obtaining a sample of human chondrocytes comprising a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) subjecting the sample of human chondrocytes derived from (a) to a treatment with a hypotonic solution, said hypotonic solution being ammonium chloride potassium lysis buffer (ACK buffer); which may be repeated multiple times, alone or in combination with other treatments.Embodiment 9B: The method of any one of embodiments 1B-8B, wherein said treatment induces cell swelling.Embodiment 10B: The method of any one of embodiments 1B-9B, wherein said sample of human chondrocytes is a sample of non-articular chondrocytes.Embodiment 11B: The method of any one of embodiments 1B-10B, wherein said sample of human chondrocytes is provided from a portion of a rib.Embodiment 12B: The method of embodiment 11B, wherein said chondrocytes provided from said portion of the rib comprise non-articular chondrocytes. Embodiment 13B: The method of any one of embodiments 1B-12B, wherein after (b), the sample of human chondrocytes has a higher percentage of non-pre-apoptotic cells compared to the sample of human chondrocytes before (b).Embodiment 14B: The method of any one of embodiments 1B-12B, wherein after (b), the sample of chondrocytes has a lower percentage of pre-apoptotic cells compared to the sample of chondrocytes before (b).Embodiment 15B: The method of any one of embodiments 1B-14B, wherein the human chondrocytes generated after (b) are used for one or more of the following: direct use of cells; in vitro culture of cells including passaging in a monolayer or three-dimensional environment, including suspension culture; tissue engineering using scaffold-free systems including self-assembly or using scaffold-based systems including natural and synthetic materials; cell transplantation; tissue transplantation; and / or grafting.Embodiment 16B: The method of embodiment 1B, further comprising passaging the cells in a monolayer or three-dimensional environment after (b). Embodiment 17B: The method of embodiment 16B, further comprising generating neocartilage with said passaged cells.Embodiment 18B: The method of any one of embodiments 1B to 17B, wherein the human chondrocytes generated after (b), or tissue engineered / created from the human chondrocytes, are subjected to a treatment comprising one or more of the following: growth factors; cytoskeletal modifying agents; hormones; toxic compounds; molecules acting upstream of a signaling cascade; varying oxygen tension; cross-linking agents; matrix-degrading enzymes, matrix molecules; and / or mechanical stimulation.

[0226] Embodiment 1C: A method of enriching a sample of non-articular chondrocytes comprising: (a) obtaining a sample of non-articular chondrocytes comprising a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) subjecting the sample of non-articular chondrocytes derived from (a) to a treatment, which may be repeated multiple times, alone or in combination with other treatments.Embodiment 2C: A method of preparing a sample of non-articular chondrocytes comprising: (a) obtaining a sample of non-articular chondrocytes comprising a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) subjecting the sample of non-articular chondrocytes derived from (a) to a treatment, which may be repeated multiple times, alone or in combination with other treatments.Embodiment 3C: The method of embodiment 1C or embodiment 2C, wherein the treatment comprises adding a hypotonic solution. Embodiment 4C: A method of enriching a sample of non-articular chondrocytes comprising: (a) obtaining a sample of non-articular chondrocytes comprising a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) subjecting the sample of non-articular chondrocytes derived from (a) to a treatment with a hypotonic solution, which may be repeated multiple times, alone or in combination with other treatments.Embodiment 5C: A method of preparing a sample of non-articular chondrocytes comprising: (a) obtaining a sample of non-articular chondrocytes comprising a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) subjecting the sample of non-articular chondrocytes derived from (a) to a treatment with a hypotonic solution, which may be repeated multiple times, alone or in combination with other treatments.Embodiment 6C: The method of any one of embodiments 3C-5C, wherein the hypotonic solution is ammonium chloride potassium lysis buffer (ACK buffer). Embodiment 7C: A method of enriching a sample of non-articular chondrocytes comprising: (a) obtaining a sample of non-articular chondrocytes comprising a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) subjecting the sample of non-articular chondrocytes derived from (a) to a treatment with a hypotonic solution, said hypotonic solution being ammonium chloride potassium lysis buffer (ACK buffer); which may be repeated multiple times, alone or in combination with other treatments.Embodiment 8C: A method of preparing a sample of non-articular chondrocytes, comprising: (a) obtaining a sample of non-articular chondrocytes comprising a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) subjecting the sample of non-articular chondrocytes derived from (a) to a treatment with a hypotonic solution, said hypotonic solution being ammonium chloride potassium lysis buffer (ACK buffer); which may be repeated multiple times, alone or in combination with other treatments.Embodiment 9C: The method of any one of embodiments 1C-8C, wherein said treatment induces cell swelling.Embodiment 10C: The method of any one of embodiments 1C-9C, wherein said sample of non-articular chondrocytes is a sample of human non-articular chondrocytes.Embodiment 11C: The method of any one of embodiments 1C-10C, wherein said sample of non-articular chondrocytes is sourced from a portion of a rib.Embodiment 12C: The method of any one of embodiments 1C-11C, wherein after (b), said sample of non-articular chondrocytes has a higher percentage of non-pre-apoptotic cells compared to said sample of human chondrocytes prior to (b). Embodiment 13C: The method of any one of embodiments 1C-11C, wherein after (b), the sample of non-articular chondrocytes has a lower percentage of pre-apoptotic cells compared to the sample of chondrocytes before (b).Embodiment 14C: The method of any one of embodiments 1C-13C, wherein the non-articular chondrocytes generated after (b) are used for one or more of the following: direct use of cells; in vitro culture of cells including passaging in a three-dimensional environment, including monolayer or suspension culture; tissue engineering using scaffold-free systems, including self-assembly, or using scaffold-based systems including natural and synthetic materials; cell transplantation; tissue transplantation; and / or grafting.Embodiment 15C: The method of embodiment 1C, further comprising passaging the cells in a monolayer or three-dimensional environment after (b).Embodiment 16C: The method of embodiment 15C, further comprising generating neocartilage with the passaged cells.Embodiment 17C: The method of any one of embodiments 1C to 16C, wherein the non-articular chondrocytes generated after (b), or tissue engineered / created from the non-articular chondrocytes, are subjected to a treatment comprising one or more of the following: growth factors; cytoskeletal modifying agents; hormones; toxic compounds; molecules acting upstream of a signaling cascade; varying oxygen tension; cross-linking agents; matrix-degrading enzymes, matrix molecules; and / or mechanical stimulation.

[0227] Embodiment 1D: A method of enriching a sample of cells sourced from a portion of a rib, comprising: (a) obtaining a sample of cells sourced from a portion of a rib comprising a mixed population of non-pre-apoptotic cells and pre-apoptotic cells; (b) subjecting said sample of cells sourced from said portion of said rib from (a) to a treatment; which may be repeated multiple times, alone or in combination with other treatments.Embodiment 2D: A method of preparing a sample of cells sourced from a portion of a rib, comprising: (a) obtaining a sample of cells sourced from a portion of a rib comprising a mixed population of non-pre-apoptotic cells and pre-apoptotic cells; (b) subjecting said sample of cells sourced from said portion of said rib from (a) to a treatment; which may be repeated multiple times, alone or in combination with other treatments.Embodiment 3D: The method of embodiment 1D or embodiment 2D, wherein said treatment comprises adding a hypotonic solution. Embodiment 4D: A method of enriching a sample of cells sourced from a portion of a rib, comprising: (a) obtaining a sample of cells sourced from a portion of a rib comprising a mixed population of non-pre-apoptotic cells and pre-apoptotic cells; (b) subjecting the sample of cells sourced from the portion of the rib from (a) to a treatment with a hypotonic solution; which may be repeated multiple times, alone or in combination with other treatments.Embodiment 5D: A method of preparing a sample of cells sourced from a portion of a rib, comprising: (a) obtaining a sample of cells sourced from a portion of a rib comprising a mixed population of non-pre-apoptotic cells and pre-apoptotic cells; (b) subjecting the sample of cells sourced from the portion of the rib from (a) to a treatment with a hypotonic solution; which may be repeated multiple times, alone or in combination with other treatments.Embodiment 6D: The method of any one of embodiments 3D-5D, wherein the hypotonic solution is ammonium chloride potassium lysis buffer (ACK buffer).Embodiment 7D: A method of enriching a sample of cells sourced from a portion of a rib, comprising: (a) obtaining a sample of cells sourced from a portion of a rib comprising a mixed population of non-pre-apoptotic cells and pre-apoptotic cells; (b) subjecting the sample of cells sourced from the portion of the rib from (a) to a treatment with a hypotonic solution, the hypotonic solution being an ammonium chloride potassium lysis buffer (ACK buffer); which may be repeated multiple times, alone or in combination with other treatments.Embodiment 8D: A method of preparing a sample of cells sourced from a portion of a rib, comprising: (a) obtaining a sample of cells sourced from a portion of a rib comprising a mixed population of non-pre-apoptotic cells and pre-apoptotic cells; (b) subjecting the sample of cells sourced from the portion of the rib from (a) to a treatment with a hypotonic solution, the hypotonic solution being an ammonium chloride potassium lysis buffer (ACK buffer); which may be repeated multiple times, alone or in combination with other treatments.Embodiment 9D: The method of any one of embodiments 1D-8D, wherein the treatment induces cell swelling. Embodiment 10D: The method of any one of embodiments 1D-9D, wherein the sample of cells sourced from the portion of the rib are chondrocytes.Embodiment 11D: The method of any one of embodiments 1D-10D, wherein the sample of cells sourced from the portion of the rib are non-articular chondrocytes.Embodiment 12D: The method of any one of embodiments 1D-11D, wherein the sample of cells sourced from the portion of the rib are human cells.Embodiment 13D: The method of any one of embodiments 1D-12D, wherein the portion of the rib comprises chondrocytes.Embodiment 14D: The method of embodiment 13D, wherein the chondrocytes are non-articular chondrocytes.Embodiment 15D: The method of any one of embodiments 1D-14D, wherein the sample of cells sourced from the portion of the rib after (b) has a higher percentage of non-pre-apoptotic cells compared to the sample of cells sourced from the portion of the rib prior to (b).Embodiment 16D: The method of any one of embodiments 1D-14D, wherein the sample of cells sourced from the portion of the rib after (b) has a lower percentage of pre-apoptotic cells compared to the sample of cells sourced from the portion of the rib before (b).Embodiment 17D: The method of any one of embodiments 1D-16D, wherein the sample of cells sourced from the portion of the rib generated after (b) is used for one or more of the following: direct use of cells; in vitro culture of cells including passaging in a monolayer or three-dimensional environment, including suspension culture; tissue engineering using scaffold-free systems including self-assembly or using scaffold-based systems including natural and synthetic materials; cell transplantation; tissue transplantation; and / or grafting.Embodiment 18D: The method of embodiment 1D, further comprising passaging cells in a monolayer or three-dimensional environment after (b).Embodiment 19D: The method of embodiment 18D, further comprising generating neocartilage with the passaged cells. Embodiment 20D: The method of any one of embodiments 1D-19D, wherein the sample of cells provided from the portion of the rib generated after (b), or tissue engineered / created from the cells, is subjected to a treatment comprising one or more of the following: growth factors; cytoskeletal modifying agents; hormones; toxic compounds; molecules acting upstream of a signaling cascade; varying oxygen tension; cross-linking agents; matrix-degrading enzymes, matrix molecules; and / or mechanical stimulation.

[0228] Embodiment 1E: A method of enriching a sample of human non-articular chondrocytes comprising: (a) obtaining a sample of human non-articular chondrocytes comprising a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) subjecting the sample of human non-articular chondrocytes derived from (a) to a treatment, which may be repeated multiple times, alone or in combination with other treatments.Embodiment 2E: A method of preparing a sample of human non-articular chondrocytes comprising: (a) obtaining a sample of human non-articular chondrocytes comprising a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) subjecting the sample of human non-articular chondrocytes derived from (a) to a treatment, which may be repeated multiple times, alone or in combination with other treatments.Embodiment 3E: The method of embodiment 1E or embodiment 2E, wherein the treatment comprises adding a hypotonic solution. Embodiment 4E: A method of enriching a sample of human non-articular chondrocytes comprising: (a) obtaining a sample of human non-articular chondrocytes comprising a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) subjecting the sample of human non-articular chondrocytes derived from (a) to a treatment with a hypotonic solution, which may be repeated multiple times, alone or in combination with other treatments.Embodiment 5E: A method of preparing a sample of human non-articular chondrocytes comprising: (a) obtaining a sample of human non-articular chondrocytes comprising a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) subjecting the sample of human non-articular chondrocytes derived from (a) to a treatment with a hypotonic solution, which may be repeated multiple times, alone or in combination with other treatments.Embodiment 6E: The method of any one of embodiments 3E-5E, wherein the hypotonic solution is ammonium chloride potassium lysis buffer (ACK buffer). Embodiment 7E: A method of enriching a sample of human non-articular chondrocytes comprising: (a) obtaining a sample of human non-articular chondrocytes comprising a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) subjecting the sample of human non-articular chondrocytes derived from (a) to a treatment with a hypotonic solution, said hypotonic solution being ammonium chloride potassium lysis buffer (ACK buffer); which may be repeated multiple times, alone or in combination with other treatments.Embodiment 8E: A method of preparing a sample of human non-articular chondrocytes, comprising: (a) obtaining a sample of human non-articular chondrocytes comprising a mixed population of non-pre-apoptotic chondrocytes and pre-apoptotic chondrocytes; and (b) subjecting the sample of human non-articular chondrocytes derived from (a) to a treatment with a hypotonic solution, said hypotonic solution being ammonium chloride potassium lysis buffer (ACK buffer); which may be repeated multiple times, alone or in combination with other treatments.Embodiment 9E: The method of any one of embodiments 1E-8E, wherein said treatment induces cell swelling.Embodiment 10E: The method of any one of embodiments 1E-9E, wherein said sample of human non-articular chondrocytes is provided from a portion of a rib.Embodiment 11E: The method of any one of embodiments 1E-10E, wherein after (b), said sample of human non-articular chondrocytes has a higher percentage of non-pre-apoptotic cells compared to said sample of cells provided from a portion of a rib prior to (b). Embodiment 12E: The method of any one of embodiments 1E-10E, wherein after (b), the sample of human non-articular chondrocytes has a lower percentage of pre-apoptotic cells compared to the sample of cells sourced from the portion of the rib prior to (b).Embodiment 13E: The method of any one of embodiments 1E-12E, wherein the sample of human non-articular chondrocytes generated after (b) is used for one or more of the following: direct use of cells; in vitro culture of cells including passaging in a monolayer or three-dimensional environment, including suspension culture; tissue engineering using scaffold-free systems including self-assembly or using scaffold-based systems including natural and synthetic materials; cell transplantation; tissue transplantation; and / or grafting.Embodiment 14E: The method of embodiment 1E, further comprising passaging the cells in a monolayer or three-dimensional environment after (b).Embodiment 15E: The method of embodiment 14E, further comprising generating neocartilage with the passaged cells.Embodiment 16E: The method of any one of embodiments 1E to 15E, wherein the sample of human non-articular chondrocytes generated after (b), or tissue engineered / created from said cells, is subjected to a treatment comprising one or more of the following: growth factors; cytoskeletal modifying agents; hormones; toxic compounds; molecules acting upstream of a signaling cascade; varying oxygen tension; cross-linking agents; matrix-degrading enzymes, matrix molecules; and / or mechanical stimulation.

[0229] Embodiment 1F: A method of enriching a sample of human cells sourced from a portion of a rib, comprising: (a) obtaining a sample of human cells sourced from a portion of a rib comprising a mixed population of non-pre-apoptotic cells and pre-apoptotic cells; (b) subjecting said sample of human cells sourced from the portion of the rib from (a) to a treatment; which may be repeated multiple times, alone or in combination with other treatments.Embodiment 2F: A method of preparing a sample of human cells sourced from a portion of a rib, comprising: (a) obtaining a sample of human cells sourced from a portion of a rib comprising a mixed population of non-pre-apoptotic cells and pre-apoptotic cells; (b) subjecting said sample of human cells sourced from the portion of the rib from (a) to a treatment; which may be repeated multiple times, alone or in combination with other treatments.Embodiment 3F: The method of embodiment 1F or embodiment 2F, wherein said treatment comprises adding a hypotonic solution. Embodiment 4F: A method of enriching a sample of human cells sourced from a portion of a rib, comprising: (a) obtaining a sample of human cells sourced from a portion of a rib comprising a mixed population of non-pre-apoptotic cells and pre-apoptotic cells; (b) subjecting said sample of human cells sourced from the portion of the rib from (a) to a treatment with a hypotonic solution; which may be repeated multiple times, alone or in combination with other treatments.Embodiment 5F: A method of preparing a sample of human cells sourced from a portion of a rib, comprising: (a) obtaining a sample of human cells sourced from a portion of a rib comprising a mixed population of non-pre-apoptotic cells and pre-apoptotic cells; (b) subjecting said sample of human cells sourced from the portion of the rib from (a) to a treatment with a hypotonic solution; which may be repeated multiple times, alone or in combination with other treatments.Embodiment 6F: The method of any one of embodiments 3F-5F, wherein said hypotonic solution is ammonium chloride potassium lysis buffer (ACK buffer).Embodiment 7F: A method of enriching a sample of human cells sourced from a portion of a rib, comprising: (a) obtaining a sample of human cells sourced from a portion of a rib comprising a mixed population of non-pre-apoptotic cells and pre-apoptotic cells; (b) subjecting the sample of human cells sourced from the portion of the rib from (a) to a treatment with a hypotonic solution, the hypotonic solution being an ammonium chloride potassium lysis buffer (ACK buffer); which may be repeated multiple times, alone or in combination with other treatments.Embodiment 8F: A method of preparing a sample of human cells sourced from a portion of a rib, comprising: (a) obtaining a sample of human cells sourced from a portion of a rib comprising a mixed population of non-pre-apoptotic cells and pre-apoptotic cells; (b) subjecting the sample of human cells sourced from the portion of the rib from (a) to a treatment with a hypotonic solution, the hypotonic solution being an ammonium chloride potassium lysis buffer (ACK buffer); which may be repeated multiple times, alone or in combination with other treatments.Embodiment 9F: The method of any one of embodiments 1F-8F, wherein the treatment induces cell swelling. Embodiment 10F: The method of any one of embodiments 1F-9F, wherein the sample of human cells sourced from the portion of the rib are chondrocytes.Embodiment 11F: The method of any one of embodiments 1F-10F, wherein the sample of human cells sourced from the portion of the rib are non-articular chondrocytes.Embodiment 12F: The method of any one of embodiments 1F-11F, wherein the portion of the rib comprises chondrocytes.Embodiment 13F: The method of embodiment 12F, wherein the chondrocytes are non-articular chondrocytes.Embodiment 14F: The method of any one of embodiments 1F-13F, wherein after (b), the sample of human cells sourced from the portion of the rib has a higher percentage of non-pre-apoptotic cells compared to the sample of human cells sourced from the portion of the rib before (b).Embodiment 15F: The method of any one of embodiments 1F-13F, wherein after (b), the sample of human cells sourced from the portion of the rib has a lower percentage of pre-apoptotic cells compared to the sample of human cells sourced from the portion of the rib before (b).Embodiment 16F: The method of any one of embodiments 1F-15F, wherein the sample of human cells sourced from the portion of the rib generated after (b) is used for one or more of the following: direct use of cells; in vitro culture of cells including passaging in a three-dimensional environment, including monolayer or suspension culture; tissue engineering using scaffold-free systems including self-assembly or using scaffold-based systems including natural and synthetic materials; cell transplantation; tissue transplantation; and / or grafting.Embodiment 17F: The method of embodiment 1F, further comprising passaging the cells in a monolayer or three-dimensional environment after (b).Embodiment 18F: The method of embodiment 17F, further comprising generating neocartilage with the passaged cells. Embodiment 19F: The method of any one of embodiments 1F to 18F, wherein the sample of cells provided from the portion of the rib generated after (b), or tissue engineered / created from the cells, is subjected to a treatment comprising one or more of the following: growth factors; cytoskeletal modifying agents; hormones; toxic compounds; molecules acting upstream of a signaling cascade; varying oxygen tension; cross-linking agents; matrix-degrading enzymes, matrix molecules; and / or mechanical stimulation.

Claims

1. A method for enriching a sample of chondrocytes, comprising: a) obtaining said sample of chondrocytes from cartilage tissue; and b) subjecting said sample of chondrocytes derived from (a) to treatment with a hypotonic solution.

2. 2. The method of claim 1, wherein the hypotonic solution is ammonium chloride potassium lysis buffer (ACK buffer).

3. A method according to claim 1 or 2, wherein the treatment induces cell swelling.

4. The method of claim 1 , wherein the sample of chondrocytes is human chondrocytes.

5. The method of claim 1 , wherein the sample of chondrocytes is provided from a portion of a rib.

6. The method of claim 1 , wherein the sample of chondrocytes is a sample of non-articular chondrocytes.

7. The method of claim 1 , wherein the sample of chondrocytes comprises a mixed population of non-pre-apoptotic and pre-apoptotic cells.

8. 8. The method of claim 7, wherein after (b), the percentage of non-pre-apoptotic cells in the sample of chondrocytes is higher compared to said sample of chondrocytes before (b).

9. 8. The method of claim 7, wherein after (b), the sample of chondrocytes has a lower percentage of pre-apoptotic cells compared to said sample of chondrocytes before (b).

10. 10. The method of claim 1, wherein the chondrocytes generated after (b) are used for one or more of the following: direct use of cells; In vitro culture of cells, including passage in a three-dimensional environment, including monolayer or suspension culture; Tissue engineering using scaffold-free systems, including self-assembly, or using scaffold-based systems, including natural and synthetic materials; Cell transplant; tissue transplantation; and / or Grafting.

11. 10. The method of claim 1, further comprising passaging the cells in a monolayer or three-dimensional environment after (b).

12. 12. The method of claim 11, further comprising generating neocartilage with the passaged cells.

13. 10. The method of claim 1, wherein the chondrocytes generated after (b), or tissue engineered / created from said chondrocytes generated after (b), are subjected to a treatment comprising one or more of the following: Growth factors; cytoskeletal modifiers; hormone; Toxic compounds; Molecules that act upstream in signaling cascades; Various oxygen tensions; cross-linking agent; Matrix degrading enzymes, matrix molecules; and / or Mechanical stimulation.

14. 2. The processed cell sample obtained by the method of claim 1, wherein the ACK buffer comprises 154.4 mM ammonium chloride, 10 mM potassium bicarbonate, and 97.3 μM ethylenediaminetetraacetic acid (EDTA) tetrasodium salt.

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