Novel methods for the production of therapeutic mammalian cells and cellspheres and compositions thereof

Abstract

Disclosed herein are compositions, devices, methods, processes, and systems for the culture of large quantities of mammalian cells in suspension culture. Also disclosed are unique and surprising cell populations derived from the disclosed methods, processes, and systems. In many embodiments, cells are cultured in suspension in a liquid culture medium that is agitated to maintain the cells in suspension, and the agitation is increased to allow the cells to grow and divide while maintaining them in suspension to create cell spheres. The disclosed devices, methods, processes, and systems are useful for adjusting the characteristics of the resulting cells based on the characteristics of the donor subject / initial cells. The disclosed cell populations are useful for treating subjects in need thereof for various diseases and conditions using cell-based therapy.

Description

[Technical field]

[0001] The disclosed compositions, devices, processes, methods, and systems are directed to the growth of large amounts of mammalian cells in suspension, particularly for the repair of intervertebral discs.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority pursuant to 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 210,859, filed June 15, 2021, and No. 63 / 219,067, filed July 7, 2021, both of which are entitled "NOVEL METHODS FOR PRODUCTION OF THERAPEUTIC MAMMALIAN CELLS AND CELL SPHERES AND COMPOSITIONS OF SAME" and are incorporated by reference herein in their entireties. [Background technology]

[0003] Disc degeneration is a major cause of back pain, which is a driving factor in healthcare costs / expenses and patient disability worldwide. Patients who experience disc degeneration have few treatment options. If treatment is available, patients can choose to undergo a surgical procedure to remove the disc. However, this type of surgical procedure is expensive and highly specialized, limiting its availability to patients with sufficient funds and access to appropriate facilities and specialists. Furthermore, these patients may be forced to wait until their symptoms (and degeneration) are severe enough to warrant a surgical procedure. In such cases, surgical procedures have a variety of outcomes. Even when successful, spinal fusion often causes accelerated degeneration of adjacent levels. Other treatment options, such as traditional approaches involving the administration of small molecules and biological therapeutics, face significant challenges, such as efficacy and safety in clinical trials. Although treatment of disc degeneration with cell therapy may be possible, such procedures suffer from challenges faced by other cell therapeutics, such as the challenge of generating uniform cells rapidly and at large scale while simultaneously minimizing costs. Typically, such methods have not been available for the generation of cell-based therapies. Summary of the Invention [Problem to be solved by the invention]

[0004] What is needed are novel therapeutic treatments that effectively and safely address cellular diseases and injuries such as degenerative disc disease while minimizing cost. These treatments need to be manufactured on a large enough scale to support a market and reduce the overall cost of the product while maintaining or improving efficacy. [Means for solving the problem]

[0005] Disclosed herein are various methods for culturing mammalian cells, including harvesting a plurality of mammalian cells, introducing the plurality of mammalian cells into a container or vessel containing sphere culture medium to create a cell / medium mixture; agitating the cell / medium mixture at an agitation speed corresponding to a first energy level, the first energy level being sufficient to maintain greater than 90% of the cells in suspension; allowing the plurality of cells to grow, divide, and form cell spheres of a first diameter; increasing the agitation speed to a second energy level, the second energy level being higher than the first energy level and sufficient to maintain the cell spheres in suspension; and maintaining the agitation speed to allow the cell spheres to achieve a larger diameter while remaining in suspension. In some embodiments, the mammalian cells may be selected from progenitor cells, stem cells, or pluripotent cells, and may be derived from muscle, liver, heart, lung, pancreas, bone, thyroid, blood, lymph nodes, muscle, brain, spinal cord, peripheral nerve, kidney, eye, skin, blood vessel, hair follicle, amniotic membrane, chorion, umbilical cord, placenta, chondrocytes, and intervertebral disc cells. In some embodiments, increasing the agitation speed to the second energy level may be linear, non-linear, or stepwise (e.g., two or more steps) over time. In some embodiments, the sphere culture medium may lack scaffolding molecules. In some embodiments, the plurality of mammalian cells may be grown in attachment culture before introducing the plurality of mammalian cells into a container or vessel containing the sphere culture medium, and / or the cells may attach to a solid surface and double before introducing the plurality of mammalian cells into a container or vessel containing the sphere culture medium. The mammalian cells may be derived from a variety of mammalian sources and species.

[0006] Also disclosed are various methods of culturing a mammalian cell population in dynamic suspension, the methods including, on a first day, introducing the mammalian cell population into a bioreactor containing sphere culture medium to create a cell / medium mixture; agitating the cell / medium mixture at a first agitation speed; allowing the cell population in the cell / medium mixture to form cell spheres; and agitating the cell / medium mixture at a second speed, the difference between the first speed producing a first shear value less than a first maximum shear value, and the second speed producing a second shear value less than a second maximum shear value.

[0007] Disclosed herein are various methods for dynamic culturing of mammalian cell spheres, including introducing a mammalian cell population into a bioreactor containing sphere culture medium to produce a cell / medium mixture; agitating the cell / medium mixture at a first agitation speed sufficient to prevent or inhibit the cells from losing their suspension; allowing the cell population in the cell / medium mixture to form cell spheres; maintaining a majority of the cells in a suspended state; isolating and recovering the suspended cell spheres; thereby dynamically culturing the mammalian cell spheres.

[0008] Also disclosed are various methods of modifying one or more characteristics of a therapeutic cell population, the methods including isolating a population of cells from a donor tissue, the donor having a first attribute having a first attribute score and a second attribute having a second attribute score; determining desired characteristics for the therapeutic cell population; selecting a first medium parameter based on the first attribute score and / or the second attribute score; selecting process parameters based on the first attribute score and / or the second attribute score and / or the medium parameters; culturing the population of cells in suspension in a container or vessel containing a sphere culture medium; maintaining the population of cells in suspension; dividing and growing the cells to form clonal cell spheres; maintaining the population of cell spheres in suspension; isolating and recovering the suspended cell spheres having the predetermined characteristics; thereby modifying one or more characteristics of the therapeutic cell population.

[0009] Also disclosed are various mammalian cell populations, including intervertebral disc cells, in which greater than 90% of the cells are negative for surface markers selected from CD24, HLA-DR / DP / DQ, CD45, CD40, CD271, CD80, CD86, or a combination thereof; and positive for surface markers selected from CD44, CD73, CD90, HLA-ABC, or a combination thereof.

[0010] Also disclosed is a mammalian cell population comprising intervertebral disc cells, wherein greater than 90% of the cells express one or more of aggrecan, collagen 1, collagen 2, collagen 6, collagen 14, decorin (DCN), biglycan (BGN), lumican (LUM), and fibromodulin (FMOD); anti-inflammatory effects confirmed by activated T cell assays.

[0011] The disclosed cells and methods can include allogenic, autologous, or xenogeneic cells and therapeutic methods. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows cell growth in various modalities. (Panel A) Cells grown in static suspension culture modality containing methylcellulose exhibit the desired sphere phenotype. (Panel B) Cells grown in a water wheel form sheets rather than spheres. (Panel C) Cells grown in an Erlenmeyer flask are not suspended and attach to the vessel wall. (Panel D) Cells grown in a Wave Bioreactor Bag form large aggregates rather than spheres. (Panel E) When grown in a stirred tank bioreactor (STR) with low agitation, cells form spheres, but the large spheres become too large in size and cause problems with oxygen transport and settling out of solution. (Panel F) Most cells grown in the STR with low agitation attach to the vessel surface, affecting growth kinetics. (Panel G) Cells grown in the STR with high agitation speed show some sphere growth but form mainly single cells. (Panel H) When using a gradient stirring profile with a low initial RPM and a high final RPM, Applicants grow spheres in the STR, limiting cell attachment. [Diagram 2] Figure 2 (Panel A) The CFD model shows that hydrodynamic conditions increase with different slopes and curvatures based on increasing RPM in the STR. (Panel B) By running the STR at various conditions and then modeling the aggrecan results using various hydrodynamic slopes, it can be confirmed that maximum shear rate (1 / s) has the strongest correlation between hydrodynamic conditions and aggrecan expression we have examined (p=0.012). [Diagram 3]Figure 3. (Panel A) Model of maximum shear and aggrecan (p=0.012), mean eddy current dissipation and cell settling (p=0.024), maximum shear and doubling (p=0.018), and dynamic agitation and sphere size (0.033). (Panel B) Illustration of the change in hydrodynamic environment as RPM is changed in dynamic culture conditions. The effect of hydrodynamic conditions on cells is modeled by growing cells at various RPMs and measuring the results. (Panel C) Illustration of cell volume fraction calculated from mean eddy current dissipation at a single sphere size and RPM. Graphic shows simulated position of cells that can predict cell settling (other RPMs, sphere sizes, and scales were also simulated). (Panel D) CFD is utilized to develop a dynamic agitation profile to minimize shear forces while also keeping the spheres in suspension as they grow in size. [Figure 4] Figure 4: Regression analysis of 109 0.25 L STR reactors grown under experimental design conditions. Using these multivariate models, Applicants are able to "maximize the desirability" of tuning the process to an outcome that results in optimal values ​​for all 11 of our high-risk factors, satisfying the comparability requirement. [Diagram 5] Figure 5 shows a study analyzing donor population process parameters. Panel A: Acceptable process parameter ranges for donor populations. Contour profiles created using two donor attributes and two process parameter variations show the acceptable operating range in white and quality attribute failures shaded by attribute. Four graphs show the ranges of process parameter 1 and process parameter 2 when donor attributes are fixed in the following positions: Panel B (low, low), Panel C (low, high), Panel D (high, low), and Panel E (high, high). In addition, these four graphs show the acceptable ranges of donor attributes and process variability across the donor and process ranges we considered. [Figure 6]Figure 6 (Panel A): The number of agitation steps affects process attributes including sphere size, aggrecan expression, and doubling. Panel B: The final RPM used for agitation affects process attributes including doubling, collagen 1 (by ELISA), and collagen 2 (by PCR). [Figure 7] Figure 7 (Panel A): Relationship between RPM and various CFD parameters including maximum shear. Panel B: Aggrecan ELISA predicted power (R2) per hydrodynamic condition normalized to maximum shear. Panel C: Aggrecan ELISA as a function of CFD calculated maximum shear in a 0.25L STR. [Figure 8] FIG. 8. (Panel A) Split stream growth diagram of five cell lines grown in both 0.25L STR and static suspension ("Cellstack") modalities. (Panel B) Comparable sphere growth in static suspension culture and STR modalities using gradient agitation. (Panel C) Relative sphere size, doubling, and aggrecan expression from day 2 to the end of culture in static suspension culture and 0.25L STR modalities are comparable. SDEV bars normalized to the average relative value for 0.25L STR. (Panel D) Cell identity as measured by flow cytometry is comparable between 0.25L STR and static modalities. [Figure 9]FIG. 9 shows results from a study analyzing the cells in an in vivo animal study. (Panel A) The rabbit study design begins with disc injury, discs are dosed 2 weeks later, and the study is terminated 6 weeks after dosing. (Panel B) The mean percent change in disc height index (DHI) from dosing to termination is shown with standard error. After dosing, disc height increased slightly in the sham group, more in the vehicle group, and more substantially in the cell therapy group. "*" indicates statistically significant difference from sham by LS Means Differences Student's t at a=.05. (Panel C) Disc histology shows increased height recovery and hydration (white area in the center of the red disc) in discs injected with cell therapy compared to vehicle or sham. [Figure 10] FIG. 10 (Panel A) shows that cells from a single donor were passaged and grown in two streams - half the cells were grown in a small-scale bioreactor (0.25L) and half the cells were grown in a 50L bioreactor - passage stream growth. Panel B shows comparable sphere growth observed in the small-scale and large-scale reactors. (Panel C) Relative sphere size and doublings from day 2 to the end of the culture in the 0.25L and 50L STR are comparable. (Panel D) Cell identity and purity as measured by flow cytometry are comparable between the 0.25L and 50L STRs. [Figure 11] Figure 11 shows results from flow cytometry analysis of five lots of intervertebral disc-derived cells from separate donors generated using the static flask method (red) or using the STR (blue). The graph shows counts versus forward scatter, which is proportional to cell size. Cells generated using the STR have a smaller size and more uniform profile. [Figure 12]Figure 12 shows the evaluation of ECM production from intervertebral disc-derived cells generated using different methods. Analysis included both collagen I and total collagen, aggrecan and sGAG (a side chain present in all proteoglycans), as well as a variety of non-conventional ECM molecules produced by the cells in culture. [Figure 13] Figure 13 shows the evaluation of the immunomodulatory properties of disc-derived cells. PMBCs are stained with CFSE dye and proliferation is assessed for CD4+ cells via flow cytometry (Panel A); histograms of non-proliferating and proliferating PMBCs are generated (Panel B). Panel C shows overlay histograms of PMBCs cultured with and without disc-derived cells showing inhibition of proliferation by the lower CFSE signal for disc-derived cells. The assay range is also demonstrated by histograms of unstained and CFSE-stained cells. Relative proliferation of multiple PMBC donors cultured with and without disc-derived cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Existing mammalian cell culture techniques and systems for the growth of non-adherent mammalian cells and / or cell spheres suffer from various shortcomings and are inadequate to maintain the required cell growth environment while providing a sufficient number of cells required to treat all subjects in need. For example, existing methods rely on culturing cell spheres using magnetic levitation, scaffolds, and / or viscous carriers. These techniques are unable to maintain a standard culture environment for more than a few cells produced from a single stationary culture device. In some cases, growth in stationary culture, for example with scaffolds or viscous carriers, results in a fraction of the population that occurs in adherent culture, due to the fact that it is not possible to completely prevent the recovery of cells that may have adhered to the interior surface during the culturing process. This results in a more heterogeneous population of cells, some of which have grown in adherent culture and some of which have grown as adherent cells.

[0014] To obtain sufficient numbers of cells, manufacturers must combine cells grown in separate static culture devices. Static cultures and devices therefor typically include a scaffold and / or viscous substrate, lack medium and / or cell mixing, and in most embodiments, the medium is not stirred, although the medium may be periodically replaced in static cultures. As mentioned above, the production of cells by static culture can introduce heterogeneity in the therapeutic dose, reducing the therapeutic value / potency of the heterogeneous dose. Pooling and isolation of therapeutic cells from static culture also greatly increases the time, effort, and cost of producing mammalian cells for culture, while also reducing the efficacy and potency of the produced cells. Finally, current methods for culturing cell spheres also lack methods for monitoring culture conditions (medium, gas, pH, etc.) and replenishing / replenishing medium components, so cell density is often very limited.

[0015] Disclosed herein are processes, methods, and systems for the large-scale growth of therapeutic mammalian cell populations that allow for rapid multiplicity of doses with improved uniformity. The disclosed processes, methods, and systems also allow for extended culture times, allowing for monitoring conditions and replenishing / replenishing various media components, resulting in further expansion of therapeutic cell populations, growth to higher cell densities, less manipulation / handling, and reduced risk of contamination. The disclosed methods, processes, and systems can be used to create distinct non-native cell populations from a wide range of tissues and cell types, including stem cells, progenitor cells, and pluripotent cells from brain, liver, kidney, cartilage, muscle, heart, lung, bone, blood, tendon / ligament, pancreas, thyroid, lymph node, spinal cord, peripheral nerve, eye, skin, blood vessels, hair follicle, amniotic membrane, chorion, umbilical cord, placenta, cartilage, and intervertebral disc tissues. The disclosed cell populations also have unique and beneficial characteristics that allow them to be used in research, drug development, and treatment for a variety of diseases, disorders, and conditions.

[0016] The disclosed processes, methods, systems, and cell populations can be used to treat or prevent a variety of injuries, damage, diseases, disorders, and conditions that affect a variety of cells, tissues, organs, and systems. In many embodiments, the disclosed cells can be useful in treating degenerative disc disease, and can promote preservation and / or restoration of intervertebral disc height (i.e., the distance between adjacent vertebrae), normalization of tissue structure, and the like.

[0017] Applicants developed the present process, method, and system after careful analysis and comparison of various cell culture methods and systems that can maintain cells in suspension without scaffolding molecules. Applicants discovered that large-scale growth of mammalian cell populations, especially cell types whose phenotypes depend on growth as cell spheres in the absence of attachment to a solid surface, requires specialized, altered culture conditions. These altered conditions allow (1) growth of cell spheres from single cells and (2) maintenance of the cells and cell spheres in suspension, while (3) minimizing disaggregation of the cell spheres, (4) minimizing high shear forces that can damage the cells and spheres, and (5) minimizing settling of the cell spheres, even for large cell spheres.

[0018] Applicant's methods and systems disclosed herein, in one embodiment, include the use of a stirred tank bioreactor (STR) to maintain cells in suspension. The disclosed STR culture environment supports the creation of high density populations with surprisingly enhanced characteristics compared to other methods, including those that may involve culture medium movement, such as wave rocking bioreactors, shake flasks, and flasks with internal water wheels. In most cases, a bioreactor may refer to a device or method for growing cells in which the medium is continuously stirred and / or mixed and may be replaced, replenished, etc., over the culture period. Furthermore, the method provides substantially improved production of mammalian cell spheres compared to existing methods, particularly those that rely on static culture (i.e., no mixing / movement of culture medium) and / or the use of a scaffold. In many embodiments, the disclosed methods and systems use continuous or intermittent fluid movement to maintain and promote cell sphere suspension while avoiding the use of a scaffold. In most embodiments, the disclosed methods and systems include growing cell spheres in an STR and / or STR device to minimize settling of the cell spheres. The disclosed methods and systems maintain the size, shape, and quality (e.g., potency, uniformity, etc.) of the disclosed cell spheres. This helps to maintain and promote uniformity of many of the beneficial cell quality attributes previously identified in conventional static scaffold culture methods.

[0019] As mentioned above, the applicants recognized that some aspects of existing cell sphere production methods were inappropriate for scale-up and / or mass production of therapeutic cells. Specifically, growth on scaffold materials, while producing populations of cells with beneficial characteristics, was costly, labor intensive, prone to contamination, and produced heterogeneous cell populations. Heterogeneity of cell populations was due to a variety of circumstances, such as differences in the microenvironment to which individual cell spheres were exposed in static culture, differences between populations of cells grown in separate devices and at different times (i.e., macroenvironments), changes in the culture environment within individual devices, etc. Thus, the applicants investigated whether it would be possible to grow cell spheres without the use of a scaffold while maintaining certain cell characteristics necessary for therapeutic efficacy.

[0020] Surprisingly, applicants herein show that scaffolding materials are not required for cell sphere growth, development, and maintenance and cell sphere-associated characteristics. Applicants analyzed various culture methods, such as wave reactors, shaker Erlenmeyer flasks, water wheels, and STRs, for their ability to grow and maintain mammalian cell spheres in suspension. Unfortunately, the methods examined proved inadequate to grow the desired population of cell spheres with characteristics consistent with conventional methods, and the results were uncertain. Specifically, the majority of cells cultured from these methods either attached to solid surfaces within the device, grew as large clumps or sheets of cells (rather than spheres), or remained as single cells.

[0021] To facilitate the development of the disclosed methods, processes, and systems, applicants have used computational fluid dynamics (CFD) to explore, analyze, and compare various culture methods capable of supporting dynamic flow of mammalian cells and culture media. CFD uses fluid mechanics to understand the flow of fluids in a given system.

[0022] After analyzing various dynamic culture conditions and systems as inappropriate for mammalian cell sphere growth, Applicants chose to explore modifications of stirred tank reactor conditions to minimize cell clumping, cell sheeting, and surface attachment. CFD was performed to optimize conditions for the small scale reactor and for scaling up reactor size while maintaining the desired conditions.

[0023] Results from the small-scale CFD studies were used to optimize the STR and to scale the systems, processes, and methods to larger systems and devices while maintaining desired cell characteristics. Initial small-scale studies were performed at approximately 0.25 L. These conditions were then optimized using CFD for the maintenance of selected cell characteristics identified in the scaffold-based static method. These conditions were maintained in even larger cultures, such as 50 L STR cultures, by adjusting the hydrodynamic conditions using CFD.

[0024] Applicants' growth of mammalian cells under the disclosed hydrodynamic conditions resulted in the generation of cell spheres from single cells and their progressive growth, maintaining the cell spheres in suspension while the cell number of the cell spheres increased. In addition, the disclosed methods and systems were able to produce cells with comparable and / or enhanced characteristics compared to conventional static culture methods. Furthermore, the disclosed cell populations have improved uniformity compared to other methods. In addition, the disclosed methods, processes, and systems provide for continuous monitoring of cell culture data (pH, dissolved oxygen, DO, etc.) which also allows for the maintenance of optical culture conditions throughout the growth / culture period.

[0025] Disclosed herein are novel methods and systems that allow for the expansion and growth of large amounts of mammalian cells. The disclosed cell culture processes, methods, and systems can include vigorous movement of culture medium sufficient to keep cells and / or cell spheres in suspension. In many embodiments, the disclosed culture processes, methods, and systems are useful for preventing or reducing the anchoring of cells and / or cell spheres to surfaces when the cells and / or cell spheres can grow and attach to the cell surface.

[0026] The disclosed processes, methods, and systems provide for the movement of culture medium and cells / cell spheres. In many embodiments, the disclosed methods, processes, and systems may provide for culturing the disclosed therapeutic cells in a bioreactor that may allow for the growth of cells and / or cell spheres in actively mixed or moving culture medium. In many embodiments, the movement of the culture medium may vary over the culturing period. In many embodiments, the amount of energy used to drive the movement of the culture medium may increase over time, which may be referred to as a gradient. In many embodiments, the dynamic movement of the culture medium may be referred to as culture medium agitation, and the gradient agitation may be referred to as dynamic agitation.

[0027] The disclosed methods and systems are useful for maintaining the phenotype, biomarkers, and characteristics of various cells that can be grown in suspension, such as stem and progenitor cells. In many embodiments, the disclosed methods and systems allow the growth and expansion of cells that typically grow as cell spheres or clusters, while maintaining their phenotype and characteristics. In many embodiments, the characteristics of cells and cell populations grown under the disclosed conditions and methods can be significantly enhanced compared to cells grown using other methods and processes, such as static methods and / or non-dynamic culturing.

[0028] The disclosed methods and systems are useful for the expansion and growth of large-scale suspension cultures of mammalian cells via the movement of culture media, cell spheres, and cells suspended therein. In many embodiments, the disclosed methods and systems provide for maintaining mammalian cells in suspension, i.e., both single cells, small clusters of multiple cells, and cell spheres. The disclosed methods and systems are useful for forming single cells into cell spheres while maintaining the cells and spheres in suspension. In many embodiments, the disclosed mammalian cells have phenotypes, biomarkers, and characteristics that are equivalent to or enhanced compared to cells grown in non-dynamic culture, e.g., cells grown in a solid matrix.

[0029] The disclosed methods and systems provide for the expansion and growth of multiple therapeutic cell populations. In many embodiments, the disclosed methods and systems provide therapeutic mammalian cells that display substantially homogeneous biomarkers and characteristics. In many embodiments, the disclosed therapeutic mammalian cell populations display low heterogeneity in terms of phenotype, biomarkers, and characteristics, such as gene expression, extracellular matrix production, anti-inflammatory signaling, surface marker display, sphere size, and the like.

[0030] Disclosed herein are methods and systems for the production of large numbers of homogenous populations of therapeutic mammalian cells.In many embodiments, the disclosed cells are more homogenous, effective, and potent in vitro and in vivo than similar cells produced from other methods, such as other methods that include scaffold molecules and / or do not include stirring or moving culture medium, and methods that include stirring that does not vary over time.The disclosed methods, processes, and systems can avoid or reduce the need to include solid or semi-solid scaffolds in culture medium.

[0031] The reduction or absence of scaffolding can allow for more uniform growth of the disclosed cells and can prevent or inhibit the growth of subpopulations of cells that may grow in contact with a solid surface, i.e., in attached or adherent culture. In many embodiments, the disclosed processes can result in a more homogenous population of cells in terms of size, characteristics, identity, etc. In many cases, a more homogenous population of cells can comprise a population of cells that is substantially larger than another population.

[0032] The disclosed methods and systems can produce a substantially homogenous population of cells with higher potency than other methods and less variability in, for example, one or more characteristics (e.g., one or more of size, doubling, surface marker expression, etc.). In many embodiments, mammalian cells derived from other methods can be composed of two or more subpopulations. In many embodiments, cell populations produced from other culture methods, for example culture methods in which the culture medium includes a scaffold, can be heterogeneous and include a substantial population of cells from two or more subpopulations. In some embodiments, as an example, a substantial portion of the cell population can be more than about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the total.

[0033] Novel compositions of therapeutic mammalian cell populations are disclosed herein. In many embodiments, the disclosed therapeutic cell populations have and express enhanced characteristics, biomarker expression, and other phenotypes. In many embodiments, the disclosed therapeutic cell populations exhibit and express unique desirable phenotypes that may be absent, absent, attenuated, suppressed, or masked in cell populations obtained by other culture methods, including, for example, growth on scaffold molecules, and / or culturing in semi-solid, static, and / or non-mixed culture media, and / or mixed / agitated media where agitation does not fluctuate over time.

[0034] The disclosed mammalian cells can be various cell types, including progenitor cells, stem cells, or pluripotent cells.Various types of stem cells and progenitor cells are well known in the art, for example, as described in Adv Drug Deliv Rev, vol. 60, no. 2, pp. 199-214, Jan 14, 2008.In many embodiments, the disclosed mammalian cells can be derived from or grown as cell spheres, for example cell spheres grown in suspension culture.

[0035] The disclosed cells can be mammalian cells that are useful for various cell and tissue-based therapies.In many embodiments, the disclosed therapeutic cell populations can be used for allogeneic therapy, autologous therapy, or xenogeneic therapy.The disclosed cells, compositions, and methods can be useful for treating one or more degenerative diseases.

[0036] The disclosed therapeutic mammalian cells can be expanded and modified cells derived from various tissues. In some embodiments, the cells are derived from human nucleus pulposus cells and tissues and can be used to treat degenerative intervertebral disc disease. In many embodiments, the intervertebral disc can be injured, diseased, or degenerated, or at risk of being injured. In many embodiments, the disclosed cells can be used to treat subjects with painful intervertebral disc or discs, for example, painful intervertebral discs in the lumbar, thoracic, and cervical regions. In many embodiments, the disclosed cells can be used to prevent pain, injury, disease, or degeneration of intervertebral discs where pain, injury, disease, or degeneration is imminent or anticipated.

[0037] One embodiment of the disclosed process, method, and system provides for the production of therapeutic populations of mammalian cells, such as nucleus pulposus cells and annulus fibrosus cells, derived from intervertebral disc tissue. In many embodiments, the cells can be derived from allogeneic human donors. In many embodiments, the disclosed therapeutic cell populations have one or more modified characteristics that make them well suited for treating and preventing various intervertebral disc disorders, conditions, and diseases, such as degenerative disc disease.

[0038] cell The disclosed methods and systems may be useful for improving the growth, uniformity, and efficacy of cells from various mammalian tissues. The disclosed cells may be isolated from various mammalian sources. In many embodiments, the disclosed cells are derived from various mammalian tissues. In many embodiments, the disclosed mammalian tissues may include one or more of neurological tissue, immunological tissue, muscle, bone, blood, cartilage, and the like. In many embodiments, the cells may be derived from an intervertebral disc, annulus pulposus, nucleus pulposus, heart, liver, kidney, lung, pancreas, articular cartilage, bone, thymus, thyroid, blood, brain, spinal cord, peripheral nerve, eye, skin, blood vessel, hair follicle, amniotic membrane, villi, umbilical cord, placenta, cartilage, or lymph node. In some embodiments, the cells are derived from cartilage, tendon, or ligament. In some embodiments, the disclosed cells are derived from an intervertebral disc tissue. In these embodiments, the cells may be derived from annulus pulposus and / or nucleus pulposus. In many embodiments, the disclosed cells are derived from nucleus pulposus.

[0039] Donor Attributes The disclosed therapeutic mammalian cells may be derived from a variety of sources. In one embodiment, the cells are derived from a human donor. Donors may exhibit and be scored based on a variety of attributes including age, sex, weight, height, body mass index (BMI), health status, and the like. In many embodiments, donors may be between about 16-65 years of age, have a body mass index between about 0-50, a weight between about 75-600 lb, and a height between about 4'5" and 7'7". In many embodiments, donor attributes may include the time from donor death to processing of the cells, e.g., between about 0-36 hours. In some embodiments, donor attributes may include health history, e.g., smoking (packs / day and duration), disease history, e.g., history of diabetes, cancer, systemic disease, and other relevant medical information. In some embodiments, donor attributes may describe the health of the dissected tissue, for example, a disc score of 1-5 (5 being very fibrous, dry, and bumpy in appearance), which captures the level of annulus fibrosis observed in the dissected tissue, and the amount of tissue in grams (1-50 grams).

[0040] Culture vessels for suspension growth The disclosed cells can be grown in suspension in a variety of vessels or containers. In many embodiments, the vessels can be configured to hold more than 1 liter of culture medium, such as 0.1L, 0.25L, 1L, 2L, 3L, 4L, 5L, 6L, 7L, 8L, 9L, 10L, 20L, 30L, 40L, 50L, 60L, 70L, 80L, 90L, 100L, 150L, ​​200L, 300L, or 400L, and less than about 500L, 400L, 300L, 200L, 150L, ​​100L, 90L, 70L, 60L, 50L, 40L, 30L, 20L, 10L, 9L, 8L, 7L, 6L, 5L, 4L, 3L, 2L, 1L, 0.25L, or 0.1L. In many embodiments, the disclosed vessel or container may be referred to as a bioreactor. In many embodiments, the bioreactor device may include one or more mechanisms for inducing or maintaining the movement of the culture medium. In many embodiments, the disclosed bioreactor may include one or more impellers, propellers, or similar devices that may facilitate the movement of the culture medium. The speed at which the medium is moved / agitated in the bioreactor may be a function of the energy used to drive the impeller. In some embodiments, the power may increase over time, for example, in one or more steps or as a linear function of time. In many embodiments, the linear function may have one or more plateaus where the force may not increase, for example, at the beginning, end, or middle of the culture period. In some embodiments, the steps may be connected by a linear or gradual increase in force from a lower step to a higher step.

[0041] The vessel or container can be formed from many materials. In some embodiments, the inner surface of the vessel or container can be coated. In some embodiments, the inner surface can be a material or can include a coating that can prevent or reduce adherence of the disclosed mammalian cells to the surface. In some embodiments, the container can include a flexible vessel, such as a bag, for growth of the disclosed cells. In these embodiments, the bag can include a surface modification to prevent or reduce cell adhesion.

[0042] The disclosed vessels or containers can be configured to promote mixing of the culture medium. In many embodiments, the vessels or containers are configured to minimize area or volume, where movement of the culture medium can be reduced sufficiently to allow cells or spheres to settle to the bottom or adhere to the interior surface of the vessel or container.

[0043] Mixing Although various methods are available for large-scale production of mammalian cells using mixing and / or movement of cell culture media, existing methods and conditions are unable to produce cell spheres and maintain the cell spheres in suspension.

[0044] Applicants herein disclose a method for the growth and maintenance of cell spheres in suspension for the creation of therapeutic cell populations with substantially homogenous characteristics. Disclosed herein are containers and vessels that may include one or more mechanisms useful for moving, stirring, and / or mixing liquids within the vessel, such as the disclosed culture medium. In many embodiments, the disclosed mechanisms are impellers, propellers, or similar devices, and the energy of mixing is adjusted to (1) support the growth and development of cell spheres, (2) prevent cells and cell spheres from settling out of suspension, and (3) minimize or prevent adhesion of cells to interior surfaces. In many embodiments, the mixing or stirring mechanism can be controlled to allow one or more speeds of culture medium movement to be altered and / or selected.

[0045] Applicants disclose herein a method for the growth and maintenance of cell spheres in suspension, where growth conditions are optimized using computational fluid dynamics or CFD. In many embodiments, CFD can be used to select growth conditions including agitation, medium, propeller / impeller speed and shape, vessel configuration, vessel volume, etc., to achieve one or more cellular characteristics, such as cell and sphere size, biomarker expression, doubling number and time, etc.

[0046] The stirring or mixing speed of the present disclosure may be varied over time to promote cell and / or sphere growth. In most embodiments, the stirring speed may be increased during the culture and growth of the disclosed cells. In many embodiments, the selection of the stirring speed may be based on the growth rate, doubling number, doubling rate, rate of change of sphere size, sphere size, biomarker production, and / or expression level of one or more biomarkers.

[0047] The disclosed stirring or mixing speed may be increased over time to improve cell sphere growth, cell sphere stability, maintain cell sphere suspension, and / or minimize cell sphere settling to the bottom of the container, and / or cell or cell sphere adhesion to the container surface. In most embodiments, the stirring speed may be maintained below maximum shear and above a speed that provides a Reynolds number of 2500 or more. Reynolds number, as used herein, refers to a unitless value based on the density of a fluid, its flow speed, dynamic viscosity, and characteristic linear dimension. Most systems are sufficiently turbulent at a Reynolds number of about 10,000.

[0048] The disclosed agitation speeds vary over the duration of the culture of the disclosed cells. In many embodiments, the starting agitation speed / energy can be selected based on one or more parameters selected from shear rate, volume average velocity, power per unit volume, and energy dissipation. In many embodiments, the starting agitation speed / energy can be selected to maintain single cells in suspension while allowing cell spheres to form and grow. In embodiments in which an impeller or propeller is used to mix and / or stir the culture medium, the agitation speed can also be selected based on the impeller tip speed.

[0049] Various parameters can be used to aid in the selection of the impeller speed. In many embodiments, the parameters include the average energy dissipation (in one example, 1×10 -6 ~1×10 -4 m 2 / s 3 ), power per unit capacity (for example, 0.05 to 130 W / m 3), maximum shear rate (in one example, 500-10,000 1 / s), average shear rate (1 / s), volumetric average velocity (in one example, 0.001-0.2 m / s), maximum velocity (in one example, 0.05-1.5 m / s), vortex size (in one example, Kolmogorov length, 120-20 μm), volumetric average shear (in one example, 0.1-25), volumetric average energy dissipation (in one example, 1×10 -6 ~1×10 -2 ), tip speed (in one example, 0.1-2 m / s), and revolutions per minute (RPM) of about 10-1000. In some embodiments, the average energy dissipation is about 1×10 -7 , 1×10 -6 , 1×10 -5 , 1×10 -4 , or 1 × 10 -3 m 2 / s 3 Super 1×10 -2 , 1×10 -3 , 1×10 -4 , 1×10 -5 , or 1 × 10 -6 m 2 / s 3 In some embodiments, the power per unit volume is about 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 W / m 3 Greater than and about 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 1.0, 0.5, 0.1, or 0.05 W / m 3 In some embodiments, the maximum shear rate can be less than about 500, 1,000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, or 8500 s -1 Greater than and about 10,000, 9500, 9000, 8500, 8000, 7500, 7000, 6500, 6000, 5500, 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1500, or 1000s -1In some embodiments, the number of stimuli is less than about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 3000, 4000, or 4500 s. -1 Greater than and about 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, or 50s -1In some embodiments, the volume-average velocity is greater than about 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, or 0.15 m / s and less than about 0.2, 0.15, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, 0.001, or 0.0005 m / s. In some embodiments, the maximum velocity is greater than about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9 m / s and less than about 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, and 0.002 m / s. In some embodiments, the vortex size is greater than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 110 μm and less than about 150, 130, 120, 110, 100, 95, 90, 85, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, or 25 μm. In some embodiments, the volume average shear is about 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.8, 2.7, or 2.9. 0.9 and less than about 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05. In some embodiments, the volumetric average energy dissipation is greater than about 1×E-6, 1×E-5, 1×E-4, 1×E-3, or 1×E-2 and less than about 1×E-1, 1×E-2, 1×E-3, 1×E-4, or 1×E-5.In some embodiments, the tip speed is greater than about 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.18, 0.17, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25 m / s and less than about 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, or 0.06 m / s. In some embodiments, the starting or ending RPM of the impeller is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 90 Greater than 0, 1000 RPM and less than about 1250, 1000, 900, 800, 700, 600, 500, 400, 300, 350, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, or 20 RPM.

[0050] As described elsewhere, various parameters may be increased / changed during the suspension culture period to maintain or optimize and / or tailor the performance characteristics / attributes of the resulting cell population. In many embodiments, the amount of energy applied to mix the culture (e.g., impeller or mixer speed) may be increased over the course of the culture period. In many embodiments, the impeller speed may be increased at smaller increments over time, which may also result in an increase in shear, average shear, and / or maximum shear over time. In one embodiment, the maximum shear is initially about 1000-3000 s -1 It increases over time, but is about 4000 to 8000 s -1 In one example, the initial maximum shear may have a final value between about 2863 s -1 On the fourth day of suspension culture, the -1 may be increased to

[0051] The disclosed agitation speeds may be increased during the culture period or culturing period. The culture period may refer to the time from inoculation of the culture medium with a number of mammalian cells (single cells and / or cell spheres) to the time the cell spheres are harvested. In many embodiments, the culture medium may be inoculated with single cells or small cell spheres (between 2-50 cells). In many embodiments, the culture period may end with harvesting of the cells, which may be primarily cell spheres between about 50-300 microns in size. In most embodiments, the increase in agitation speed may be constant and substantially linear, or the increase may be stepwise, e.g., may include 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more steps between inoculation and harvesting. In many embodiments, the disclosed methods may include three distinct steps of agitation speed over the culture period, i.e., an initial setting when the culture is inoculated, a second setting, and a third setting, followed by harvesting of the cell spheres. In one embodiment, for example, if the culture vessel is 200-300 ml, the disclosed methods may include starting and ending RPMs of 75-275 RPM for a 250 mL vessel.

[0052] The initial agitation speed can be selected to both maintain the cells in suspension and allow for the formation of cell spheres - i.e., avoiding forces sufficient to break up or disrupt the cell spheres.

[0053] Culture medium Other process parameters The disclosed cells can be grown in suspension culture and various growth parameters adjusted. In some embodiments, the seeding density is about 100 to about 1 x 10 6The number of cells / mL may vary. The vessel and growth conditions, such as culture time, temperature, volume, dissolved oxygen, pH, medium exchange rate, perfusion supplement exchange rate, etc., may also vary. In various embodiments, the culture time may vary from about 7 to about 25 days. In some embodiments, the final cell density may be about 100 to about 10e6 cells / mL. In various embodiments, the culture temperature may vary from about 35 to about 38°C). The volume of medium in the vessel may also vary from about 50 to about 100%, the dissolved oxygen percentage may vary from about 50 to about 110%, and the pH of the medium may vary between about 7.2 to about 7.8. In various embodiments, the medium and perfusion exchange rate may vary from about 0 to about 500% / day.

[0054] Cell growth / doubling The disclosed methods, processes, and systems are useful for improving the growth rate and / or expansion potential of mammalian cells grown in vitro without loss of potency. In many embodiments, the disclosed methods, processes, and systems allow for more cell doublings without manual intervention (i.e., "passaging" cells from one culture vessel to two, dissociating cell spheres into single cells and re-growing more spheres, etc.). In many embodiments, the disclosed methods, processes, and systems allow for faster growth of more uniform cells and more homogenous cell populations.

[0055] The disclosed cells may be grown in adherent culture before or after growth in suspension.In many embodiments, the disclosed cells may undergo two or more divisions before being placed in suspension.In many embodiments, the population of the disclosed cells may undergo 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more "doublings" before being transferred to suspension culture, where "doubling" refers to a doubling or 2x increase in the population of cells.

[0056] When grown in suspension, the disclosed cell populations may double between 1 and 15. In many embodiments, the disclosed cells may undergo more than one doubling during growth in suspension, e.g., greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 "doublings" before being harvested.

[0057] As used herein, a cell population may be described as undergoing one or more doublings, in which the number of cells in the population is "doubled" - thus, a population that has undergone two "doublings" has increased in number four-fold, or 4x, from the initial inoculation into culture, where x is the starting number of cells.

[0058] Sphere Size The disclosed methods, processes, and systems provide for the growth of mammalian cell spheres. In many embodiments, the disclosed cell spheres are between about 20 μm and 200 μm in size. In many embodiments, the mean average size of the cell spheres produced by the disclosed methods is between about 50 μm and about 75 μm. In many embodiments, about 80% or more of the disclosed cell spheres are between about 93 μm and 164 μm. In many embodiments, the cell spheres can include a plurality of cells, e.g., a clonal population, derived from a progenitor cell. In many embodiments, the cell spheres may comprise more than about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 4000, 450, or 500 cells and / or less than about 2000, 1500, 1000, 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 cells.

[0059] Biomarkers Applicant's disclosed methods and systems may induce and / or support various gene and / or protein expression profiles. In many embodiments, the gene and protein expression profiles of the disclosed cells may promote tissue repair or support health. In some embodiments, the expressed genes and proteins may promote repair and / or maintenance of intervertebral discs, such as discs at risk for or exhibiting disc degeneration. In many embodiments, this difference in gene expression may be different from that produced by other culture methods, such as culture methods that include one or more viscous scaffolds to keep cells in suspension and may use one or more techniques for medium agitation, such as waterwheels or movement of culture or container devices. In some embodiments, expression is enhanced for genes and proteins shown to be suppressed in disc degeneration.

[0060] Biomarkers, including genes, proteins, and compounds, may exhibit enhanced expression as a result of the method. The disclosed biomarkers may include one or more biomarkers related to proteoglycans or collagen. In many embodiments, the disclosed biomarkers may relate to one or more small leucine-rich proteoglycans. The disclosed genes, proteins, or compounds may be selected from one or more of nucleus pulposus markers, extracellular matrix molecules, glycosaminoglycans (GAGs), small leucine-rich proteoglycans (SLRPs), aggrecans (NP_001126, XP_001131727, XP_001131734; NP_037359; NP_001356197, XP_006720482; XP_011519615; XP_011519616), collagen 1, 2, 6, 12, and the like. In many embodiments, SLRPs are glycoproteins including decorin (DCN; NP_001911; NP_598010; NP_598011; NP_598012; NP_598013; NP_598014), biglycan (BGN; NP_001702), lumican (LUM; NP_002336), and fibromodulin (FMOD; NM_002023.5).

[0061] Expression Biomarker expression can be measured by a variety of methods. In many embodiments, biomarker expression is measured by one or more of flow cytometry, PCR, RT-PCR, protein assays, glycan assays, ELISA assays, colorimetric assays, and the like. In one embodiment, flow cytometry can be performed with a fluorochrome-conjugated mouse anti-human monoclonal antibody. In many embodiments, flow cytometry can include one or more isotype controls. In many embodiments, the disclosed cells can be incubated with one or more antibodies that recognize surface receptors, markers, or proteins. In many embodiments, incubation can be performed for about 20-90 minutes at 4° C. in the presence of serum albumin and human Fc block. Various surface markers can be assayed, such as one or more of HLA-DR / DP / DQ, CD24, CD44, CD73, CD90, HLA-ABC, CD34, CD45, CD40, CD271, CD80, Gd2, Flt-1, and CD86. In some embodiments, dead or metabolically inactive cells can be identified and then excluded from the analysis, for example, using a compound that helps distinguish these cells from live cells (e.g., the compound 7-AAD). In many embodiments, biomarker expression, e.g., surface marker expression, can be quantified by flow cytometry and data on expression across a population, where less than 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% and greater than about 0.1%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the population expresses the biomarker.

[0062] The term "about" or "approximately" refers to the tolerance of a particular value as determined by one of ordinary skill in the art, which depends, in part, on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. Whenever the term "about" or "approximately" precedes a first number in a series of two or more numbers, it is understood that the term "about" or "approximately" applies to each and every one of the numbers in the series. In many embodiments, "substantially" or "substantial" may refer to a majority of a portion, such as greater than about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98%, or 99% of a set or population. In some cases, substantial may be used in the context of an improvement, and in these cases the improvement may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or 300% or more. EXAMPLES

[0063] Examination of scaffold-free culture methods Intervertebral disc tissue obtained from a recently deceased mammalian donor subject. Individual nucleus pulposus cells were isolated from the intervertebral disc tissue via enzymatic digestion. Single cells were then expanded in adherent culture. Cells were then passaged into separate groups, with each cell group grown in one type of non-adhesive or attachment-independent culture condition. Specifically, one cell group was grown by the previous method, containing a viscous scaffold in a flask. Other groups were grown in rocking non-stick bags, rocking Erlenmeyer flasks, vessels containing an internal rotating wheel, and STR-based culture. Each cell group was seeded in the same culture medium cocktail used in the previous flask system, except for the control flask group, where the medium lacked methylcellulose.

[0064] Figure 1 shows images of various flasks / vessels / containers / bioreactors and systems. Additionally, Figure 1 shows microscopic images of one embodiment of the disclosed cells / spheres obtained from the method. These microscopic pictures were made at various magnifications just before harvesting.

[0065] At harvest, cell spheres were dissociated into single cells and counted (using a K2 automated cell counter; Nexcellom) to obtain a final total cell number. The number of cell doublings was calculated for each modality (doublings = 3.32 (log(harvesting number)-log(inoculation number)) and recorded. Recorded doubling values ​​were compared to cell doublings in the flask system.

[0066] After initial analysis, applicants identified methods that supported cell growth and sphere formation without significant cell attachment to the vessel surface. Cells from these methods were then further processed (washed, concentrated, and frozen at -196°C) for additional testing.

[0067] Cells were grown in four modalities with passive control and one with active control. The first modality, and control condition, was static suspension culture in CellSTACK (Corning) with ultra-low adhesion coating (n=8). In this static culture, the medium contained 0.75% methylcellulose (Benecel A4M, Ashland) to provide a scaffold, prevent cell adhesion, and promote sphere growth. In the remaining modalities, cells were suspended in methylcellulose-free medium with fluid movement. The second modality was a 500 ml unbaffled glass Erlenmeyer flask with a vented cap (Chemglass, n=2 at 100 and 130 revolutions per minute (RPM)). Flasks were siliconized prior to use (Sigmacote siliconization reagent, Sigma-Aldrich) to reduce cell adhesion to the vessel wall. The third modality was a vented cap water wheel (PBS 0.1MAG, PBS Biotech, n=2). The fourth modality was a wave bioreactor (Xuri Cell Expansion System, Cytiva, n=2). The reactor was shaken at a maximum of 4 oscillations per minute with a shaking angle of 4 degrees. In each passive control modality, temperature was maintained at 37°C. Dissolved oxygen and pH were maintained by a vented cap in 95% air and 5% CO2 using an incubator.

[0068] The fifth modality utilized active control, where cells were grown in 0.25 L glass stirred tank bioreactors (0.25 L DASbox Mini Reactor Systems, Eppendorf). STR vessels were siliconized with Sigmacote prior to use to reduce cell adhesion to the vessel walls. Three agitation speeds (low, medium, high) were utilized using an 8-blade impeller (n=2 per condition). Dissolved oxygen and pH were actively controlled using a mixture of CO2, air, O2, and N2 gas overlay.

[0069] Cells were grown for comparable periods in each of the five modalities. Macroscopic images of the flasks / bioreactors and microscopic images of the cells / spheres were obtained at various magnifications immediately prior to harvest.

[0070] [Table 1]

[0071] Mixing modeling research The 0.25L STR was run in 22 different conditions, including 6 conditions with various static agitation profiles and 16 conditions with gradient agitation profiles. Cell doublings, sphere size, and extracellular aggrecan matrix production were measured for each condition. CFD was then used to generate hydrodynamic models of the hydrodynamic forces in the 0.25L STR (described below). These hydrodynamic models were compared to cell quality output using standard least squares regression. Finally, the STR parameters were modified to the new optimal set points identified using the regression models of hydrodynamic forces and cell quality.

[0072] cell doubling At harvest, cells were dissociated from the spheres and final cell counts were obtained using a K2 automated cell counter (Nexcelom) to assess overall growth kinetics. Cell doublings (doublings = 3.32 (log(harvest number)-log(inoculation number)) were reported as fold doublings compared to those found in static suspension cultures.

[0073] Sphere Size Sphere size was measured via image analysis in ImageJ software

[22] . Three pictures of the concentrated sphere collection per container were taken at 40x magnification and analyzed using a custom ImageJ plugin written in-house. For each picture, the custom plugin created an 8-bit grayscale image, removed outliers, filled holes, and used a watershed to identify spheres and segment overlapping spheres. Finally, the plugin set scales and measurements to capture sphere feature data and adjusted image magnification, after which the "Analyze Particles" function captured these measurements for all spheres with sizes between 1000 and 7500 pixels and circularity between 0.15 and 1.0.

[0074] Extracellular matrix protein in vitro culture assay Intervertebral disc-derived cells were plated in 96-well round-bottom ultra-low attachment plates at 2.5 × 10 in DMEM / F12 containing 0.5% fetal bovine serum and 50 ug / mL gentamicin. 5 Cells were seeded at 1000 x g / well. Cell cultures were incubated at 37° C., 5% CO2 for 72 hours. Supernatants were removed from the cultures for analysis by ELISA assay to determine the concentration of aggrecan (Aggrecan (PG) Human ELISA Kit, Thermo Fisher Scientific). An internal reference control cell line was run for each assay to verify assay performance in parallel.

[0075] Computational Fluid Dynamics Modeling Detailed models of two STR systems (0.25L DASbox by Eppendorf and HyPerforma 5:1 50L SUB by Thermo Fisher Scientific) were then created using DesignModeler, Meshing, Fluent&CFD Post (ANSYS18.1, Ansys). The systems were modeled using impeller RPM and seven agitation speeds intended to capture the magnitude of the hydrodynamic conditions. The average energy dissipation (m 2 / s 3 ), power per unit capacity (W / m 3 ), maximum shear rate (1 / s), average shear rate (1 / s), volume average velocity (m / s) and tip speed (m / s) were calculated using a set of assumptions and equations found in the Supplementary Material.

[0076] Cell doublings, sphere size, and aggrecan production from 22 STR vessels were modeled using these CFD-derived hydrodynamic conditions and compared to empirical findings using standard least squares regression. CFD was also used to calculate the correlation between mean eddy turbulence dissipation and the distribution of cells in each part of the reactor (cell volume fraction), assuming that the spheres were the maximum observed sphere size from STR runs.

[0077] Comparison of static suspension and STR modalities STR parameters were updated based on agitation and CFD regression models to optimize doubling, sphere size, and aggrecan production values. The static suspension process and the new STR culture process were compared using cells from five separate human donors cultured in parallel. A gradient agitation profile was used for the STR, where the agitation rate was increased over time, which increased the sphere size. One replicate for the static suspension condition and three replicates for the 0.25L STR were performed. Cells generated from each were compared for key attributes including cell doubling, sphere size, aggrecan expression, and flow cytometry identity. Cell bioactivity was also compared in an in vivo rabbit model of intervertebral disc degeneration.

[0078] Update on extracellular matrix protein in vitro culture assay For this comparison between static suspension and 0.25L STR studies, the assay was slightly modified to improve sensitivity. Intervertebral disc-derived cells were cultured at 2.5 x 10 in DMEM high glucose with pyruvate supplemented with 1x ITS + premix, 0.35 mM L-proline, 0.17 mM 2-phospho-L-ascorbic acid, and 50ug / mL gentamicin. 5 Cells / well were seeded into 96-well v-bottom polypropylene plates. Cell cultures were incubated at 37° C., 5% CO2 for 5 days. Supernatants were removed from the cultures for analysis by ELISA assays to determine the concentrations of aggrecan (Aggrecan (PG) Human ELISA Kit, Thermo Fisher Scientific) and collagen I (Abcam). An internal reference control was run for each assay to verify assay performance.

[0079] Identity by flow cytometry Cell identity was measured using flow cytometry with fluorochrome-conjugated mouse anti-human monoclonal antibodies, including appropriate isotype controls. Cells were incubated with antibodies for 30-60 min at 4°C in PBS containing 0.5% human serum albumin, human Fc block, and the following antibodies: HLA-DR / DP / DQ, CD24, CD44, CD73, CD90, HLA-ABC, CD34, CD45, CD40, CD271, CD80, and CD86 (BD Biosciences, San Jose, CA, USA). Forward scatter histograms were generated. Positive expression was assessed in the live cell population using 7-AAD (BD Biosciences) staining to exclude dead cells. Flow cytometry measurements were performed on a CytoFLEX flow cytometer (Beckman Coulter Life Sciences, Indianapolis, IN USA) and analyzed using FlowJo software (BD Bioscience). More than 10,000 events were collected for each analysis.

[0080] Process Tunable Modeling Five donors were selected to represent our donor population, with four donors (Donors 1-4) having donor quality attributes at either end of our acceptable range, and one donor (Donor 5) having donor quality attributes in the center of the range. A data table of donor attributes was created to provide the covariate data within the DOE dialog (Table 2), where the lower end of the range is represented as -1, the upper end of the range is selected as +1, and the midpoint of the range is represented as 0. By creating this covariate table, the disclosed data analysis software (JMP from SAS, as an example) automatically creates a Whole Plot with the Random Block Role of each donor in the DOE in addition to the model role selected.

[0081] [Table 2]

[0082] A D-optimal DOE (design of experiments) was created using two covariate donor quality parameters, four high-risk medium parameters, and five high-risk process parameters (Table 3). Medium and process setpoints were given a continuous role in the DOE dialog. The DOE was designed with six runs of the 14 STR conditions in a blocking role. Main effects, interaction effects, and curvature estimability were each set to "Necessary" in the DOE dialog. A D-optimal DOE was created and run. A seventh run was run without the DOE dialog, where each of the five cell lines was run in triplicate under center-point conditions for medium and process parameters. If the experimental conditions were not maintained at the DOE setpoints, an additional STR was run as the eighth run. The seventh and eighth runs were each assigned their own blocking level.

[0083] [Table 3]

[0084] Data from the eight trials was analyzed using regression and ANN techniques (outlined below). Using both methods, optimal conditions were identified for all eleven donors, media, and process set points. To test these set points, the STR was run in triplicate at our process center points and at the optimal conditions identified by the ANN and regression models.

[0085] DOE was first assessed for normal distribution using Anderson-Darling goodness-of-fit test. The normally distributed data was then analyzed by standard least squares regression with restricted maximum likelihood. All main, interaction, and quadratic effects were included in the initial model. Inputs that did not contribute significantly to the model (p>0.05) or did not have higher order effects were still included in the model and were removed from the model one by one in descending order of p-value. Once only significant effects remained, adjusted R-squared and analysis of variance were used to assay model quality. To find optimal process settings, a predictive profiler, for example a predictive profiler in the analysis software, was used to maximize cell doublings. The profiler can help visualize the response surface when one or two input parameters / factors are changed. In many cases, the profile can be a cross-section of the data that allows exploration of various spaces, such as opportunity spaces. Potential multivariate ranges were assessed using the contour profiler available in the JMP software.

[0086] Results from the sustainable DOE demonstrate that by adjusting / selecting process and media parameters, donor cells with various attributes can be generated with specific performance characteristics. In many embodiments, the performance attributes can be selected based on various criteria or the intended use of the cells.

[0087] In vivo assessment of bioactivity For in vivo evaluation of bioactivity, female New Zealand white rabbits (3-4 kg) were used under approval by a private Institutional Animal Care and Use Committee (IACUC). Fourteen rabbits were anesthetized and the lumbar region of the spine was surgically exposed. Three lumbar intervertebral discs (L3-L4, L4-L5, L5-L6) were injured via insertion of an 18-gauge needle 5 mm into the disc. L2-L3 was left uninvolved as a healthy control. Muscles and skin were then closed using sutures and animals were monitored during recovery. Two weeks later, rabbits were prepared again for surgery and 0 or 67,000 cells in 25 ml of medium from two different donors were injected into the discs via a 27-gauge needle (n=3-6 / condition in two animals per condition). Sham surgery was also performed for comparison (n=3). Half the cells were grown in static culture containing methylcellulose and mixed with conventional media (1% sodium hyaluronate containing Profreeze (Lonza Bioscience), dimethyl sulfoxide, saline, and human serum albumin), while the other half of the cells were grown in STR and mixed with updated media that omitted Profreeze.

[0088] X-ray images of the lumbar spine were obtained every 2 weeks. Measurements between 18 bony landmarks were performed in a blinded manner to calculate the disc height index (DHI) in various conditions. DHI and its measurement are well known in the art, as disclosed in LI Silverman et al., "In vitro and in vivo evaluation of discogenic cells, an investigational cell therapy for disc degeneration," Spine J, vol. 20, no. 1, pp. 138-149, Jan 2020. Animal weights and behavior were also noted for any abnormalities. After 6 weeks of dosing, animals were sacrificed and discs were explanted, fixed, decalcified, sectioned through the center of the disc, and stained with hematoxylin and eosin (H&E), Safranin O, and picrosirius red / alcian blue mixture. Slides were evaluated by a board-certified veterinary pathologist for the presence of abnormal tissue or inflammation, and any potential normalization of tissue structure.

[0089] Scaling up to large-scale STR A CFD model of the HyPerforma 5:1 50L SUB (Thermo Fisher Scientific) was generated. RPM was scaled by maintaining maximum shear rate (s-1) across multiple scales. Cells from a single donor were passaged into a 0.25L STR (DASbox, n=2) and a 50L pilot-scale commercial system (HyPerforma 5:1 50L SUB by Thermo Fisher Scientific, n=1). The resulting disc-derived cells were compared via flow cytometry for doubling, sphere size, and identity using the methods described above.

[0090] ECM production Serum-free ECM culture medium was prepared consisting of high glucose DMEM, 1x ITS+premix (corning), 0.35 mM proline, 0.17 mM ascorbic acid-2-phosphate, and 25-50 ug / mL gentamicin sulfate. Liquid nitrogen samples were thawed and transferred to centrifuge tubes containing medium. Samples were centrifuged at 200-400 x g for 5 minutes to pellet the cells. The supernatant was aspirated and the cell pellet was resuspended in 1-3 mL of ECM culture medium. Cell concentration was measured using a K2 or cellaca cellometer (Nexcelom) and the concentration of cells was adjusted accordingly to 0.5-3 x 10 6 The viable cells / mL were adjusted. 200-300 μL of each cell suspension was added to each well of an ultra-low attachment 96-well round-bottom plate. The plate with cells was incubated in a cell culture incubator (37°C, 5% CO2). After 3-5 days, the cell plate was removed and centrifuged at 200-400 x g for 5 min. The supernatant was carefully transferred to a new 96-well v-bottom plate without disturbing the cell pellet.

[0091] Collagen I ELISA assays were performed according to standard practice. Aggrecan ELISA assays were performed according to standard practice, except that 60-100 μl samples were used instead of 50 μl samples and the volume of incubation buffer was adjusted to equal the amount of input sample volume (60-100 μl). Total amounts of collagen I and aggrecan in cell supernatants were determined using standard curves generated for each analyte.

[0092] PCR assay Samples were obtained from either cell pellets after in vitro culture in ECM efficacy assays or cells obtained from suspension culture harvest before dissociation (Note: samples can also be obtained after dissociation). Fresh cell samples (i.e., obtained from cell pellets after in vitro culture) may express different biomarkers at different levels compared to cells obtained from suspension culture. In many embodiments, cell samples can be similarly processed for PCR analysis. Samples were dissolved in TRIzol reagent and stored at -80C before analysis. RNA was extracted using the PureLink RNA Microscale Kit. Gene expression for ACAN (Hs00153936_m1), COL1A2 (Hs00164099_m1) and COL2A1 (Hs01060325_g1) was measured using commercial TaqMan assays and normalized to the expression of the housekeeping gene HPRT1 (Hs02800695_m1). Reverse transcription to cDNA and PCR amplification steps were performed in a single experiment using the 1-step Fast Virus Master Mix (ThermoFisher catalog 4444436) on a QuantStudio5 real-time PCR system using the recommended cycling conditions.

[0093] Pellet weight assay Prepare serum-free pellet culture medium consisting of high glucose DMEM, 1x ITS+ premix (corning), 0.35 mM proline, 0.17 mM ascorbic acid-2-phosphate, and 25-50ug / mL gentamicin sulfate. Thaw cells and resuspend in 1-9mL pellet culture medium. Count cells in a K2 or cellaca cellometer. Transfer 3-9 million cells to a 15mL tube and adjust cell concentration to 1-2 million cells / mL. Centrifuge cells at 200-400xg for 5 minutes to pellet cells. Culture pellet in 37C and 5% oxygen incubator for 2 weeks, changing medium every 2-3 days. At the end of culture, collect cells and weigh to determine wet weight of pellet.

[0094] Biochemical assays to measure sGAG and hydroxyproline Prepare papain digestion buffer by mixing cysteine ​​hydrochloride to 0.01 M and papain to 0.125 mg / mL in basic digestion buffer (0.10 M disodium phosphate, 0.01 M ethylenediaminetetraacetic acid disodium salt dihydrate, pH 6.5). Digest the cell pellet in 200-500 μL of papain digestion buffer at 60 °C for 12-18 h. Vortex the digestion mixture to dissipate the digested pellet. Assay sGAG and total collagen according to known protocols. The collagen assay was modified from Cissell et al. Specifically, the reaction volume of Cissell was halved, and consequently the input sample volume and the volume of all solutions were halved.

[0095] Sulfated glycosaminoglycans were assayed using a method adapted from (Eur Cell Mater, 2015 Apr 19;29:224-36), except that the volume of the DMMB solution was 800 mL instead of 1 L to produce a 1.25× solution. The pH of this solution was adjusted to 1.5 with HCl on the day of use, and the solution concentration was brought to 1× with 0.03162 M HCl to maintain a pH of 1.5.

[0096] T cell immunomodulatory assay A sample of disc-derived cells from liquid nitrogen was thawed and transferred to a centrifuge tube containing 3-8 mL of DMEM / F-12 (disc-derived cell culture medium) containing 10-20% FBS and 25-50 ug / mL gentamicin sulfate. The cells were centrifuged at 200-400 x g for 5 minutes to pellet the cells. The supernatant was aspirated and the cells were resuspended in 1-3 mL of disc-derived culture medium. The cell concentration was measured using a K2 or cellaca cellometer and the cell concentration was adjusted accordingly to 0.2-2 x 10 6The cell suspension was adjusted to viable cells / mL. 200-300 μL of each cell suspension was added per well of a 96-well tissue culture plate (flat bottom). The plate was incubated in a cell culture incubator (37°C, 5% CO2) for 1-4 days.

[0097] After incubation, the PBMCs were thawed and plated on the same plates as the disc-derived cells according to the following protocol: Disc-derived cell culture medium containing mitomycin at a concentration of 30-50 μg / mL is prepared, which may be referred to as "pre-culture medium" and may be useful to inhibit or prevent proliferation. 100-200 μL of pre-culture medium containing mitomycin is added to the disc-derived cells and incubated at 37C for 1.5-3 hours. While the cells are incubating in mitomycin, PBMCs are prepared as follows: PBMC samples from liquid nitrogen were thawed and transferred to centrifuge tubes containing 3-8 mL of Immunocult XF T Cell Expansion Medium (Stem Cell Technologies) (Quench Medium) containing 10-20% FBS and 25-50 ug / mL gentamicin sulfate. The tubes containing the thawed PBMCs were then centrifuged at 200-400 x g for 5 minutes to pellet the cells. The supernatant was aspirated and the samples were resuspended in 1–3 mL of PBS. Cell concentrations were measured using a K2 or cellaca cellometer and adjusted accordingly to a cell concentration of 1–2 × 10 6The concentration was adjusted to viable cells / mL. CFSE dye was resuspended in 18-40 μL DMSO, then 0.5-1.5 μL of resuspended CFSE was added to the cells and incubated for 5-25 min with regular mixing by vortexing every 5-10 min. After CFSE staining was completed, 3-4 volumes of Quench Medium were added and the mixture was incubated for 3-8 min. PBMC samples were centrifuged at 200-400 × g for 5 min to pellet the cells. The supernatant was aspirated and the cells were resuspended in an appropriate volume of Immunocult XF T Cell Expansion Medium containing 10-20 ng / mL IL-2 and 25-50 μL gentamicin to obtain a PBMC concentration of 1-5 million cells / mL. A subset of PBMCs was activated with CD3 / CD28 activator by adding the activator to the cells at a concentration of 2-50 μL / mL. A subset of PBMCs that were not treated with activator served as non-activated controls. Activated PBMCs were added to wells with and without disc-derived cells at a density of 100,000-500,000 cells / well in 100-200 μL of medium. Non-activated control PBMCs were added at the same density to wells without disc-derived cells.

[0098] Cell cultures were incubated for 3-5 days, and then cells were prepared for flow cytometry to assess PBMC proliferation according to the following steps: 100-200 μL of TRYPLE (Thermofisher) was added to each well and incubated at 37°C until the disc-derived cells were detached (5-10 min). Cells were pipetted to mix and break up clusters and transferred to a 96-well polypropylene plate. Cell plates were centrifuged at 200-400 x g for 5 min. The supernatant was decanted and cells were resuspended in 90 μL of flow buffer (PBS + 1% HSA) containing 0.02-0.08 ug / mL human Fc block (BD Biosciences). Resuspended cells were incubated at RT for 10-15 min, after which 5-20 μL of CD4 antibody or IgG isotype control (for control samples) was added, and samples were then mixed and stained at 4C for 30-60 min. Flow buffer was added to a total volume of 250-300 μL and then centrifuged at 200-400×g for 5 min. The supernatant was decanted and 250-300 μL of flow buffer was added followed by centrifugation at 200-400×g for 5 min. The supernatant was again decanted and the cells were resuspended in 200-300 μL of flow buffer, the samples were transferred to microcentrifuge tubes and the FITC signal in CD4 positive cells was analyzed by flow cytometry. The average FITC signal of CD4+ activated PBMCs without disc-derived cells was calculated and this value was used to calculate the relative proliferation rate of activated and non-activated CD4+ PBMCs cultured with disc-derived cells using the following formula: Relative proliferation rate = 100 - 100 × ((1 / (FITC-M_sample)) / (1 / (FITC-A_control)) FITC-M sample = average FITC signal of all CD4+ cells measured in each sample FITC-A_Control = Mean FITC_M signal of all samples of activated PBMCs cultured without disc-derived cells

[0099] statistical analysis Means and standard deviations (SD) or standard errors were calculated for each set of experimental conditions. When multiple groups were compared, standard least squares regression models were constructed and pairwise comparisons of experimental groups were performed by post hoc least squares means differences student's t test at alpha = 0.05.

[0100] Computational Fluid Dynamics Methods Detailed models of the two systems (0.25L DASbox by Eppendorf and HyPerforma 50 liters by Thermo Fisher Scientific) were created using Sign Modeller, Meshing, Fluent&CFD Post by ANSYS18.1. To account for blade movement, a moving reference zone was created around the impeller blades. A "mesh" was then created for the two tank scales, dividing the computational domain into several small volumes or elements. The element dimensions were made small enough to capture the main features of the modeled process, especially in the regions of high turbulence (i.e., in the vicinity of the impeller blades). After the creation of an initial coarse mesh, the mesh was refined around the impeller blades and probes, where applicable. Initially, there were about 90,000 elements on average in the coarse mesh for each reactor. With refinement, the number rose to 4.4 million elements. Conservation equations of the transport phenomena were then applied and solved for each element. This process was performed for each vessel configuration, at each of the seven stirring speeds.

[0101] A set of assumptions and equations were used to develop the CFD model. A table of abbreviations is provided below. In most embodiments, the Reynolds number (a unitless coefficient that describes flow characteristics) is kept in the turbulent region, i.e., Reynolds number >2500 (turbulent flow).

[0102] [Table 4]

[0103] The Reynolds number (a unitless coefficient describing flow characteristics) was assumed to be in the turbulent region, i.e., Reynolds number > 2500 (turbulent flow). The Reynolds number is given by the formula:

number

[0104] The input power is, in CFD, P = πNM It can be calculated as follows.

[0105] The impeller power number Po(-) can then be calculated:

number

[0106] The shear rate calculation is specifically related to the energy dissipation in the vessel. When the energy introduced by the impeller is dissipated, this energy is consumed by nearby fluids and other particles (in this case, specifically proteins) that can be adversely affected by high rates of energy dissipation (i.e., high shear). The shear rate calculation is calculated as follows: Vessel average turbulent shear rate:

number

number

number

[0107] Selection of new culture modalities Cells were grown in static suspension culture, wave reactor, water wheel, shake Erlenmeyer flask, and 0.25L STR. Cells grown in static culture generated typical intervertebral disc-derived cell spheres (Figure 1 Panel A). Cells grown in water wheel (relative doubling 1.16, SD 0.04) formed long sheets of cells along the wheel and accumulated in the corners of the vessel (Figure 1 Panel B). Cells grown in shake Erlenmeyer flasks had comparable doublings compared to the original static control (100 RPM: 1.04 relative doubling, SD 0.09; 130 RPM: 1.07 relative doubling, SD 0.32), but no spheres were generated and cells attached to the vessel wall and formed clumps (Figure 1 Panel C). Cell aggregation was most pronounced in the wave reactor, where large aggregates of cells formed (relative doubling of 0.50, SD 0.31) (Figure 1 panel D).

[0108] In the 0.25L STR, cells grown at the low RPM setting formed spheres. However, the spheres became too large in size (Figure 1 Panel E), causing problems with the spheres settling out of solution (Figure 1 Panel F); medium and high RPM settings resulted in limited sphere formation (Figure 1 Panel G). Doublings were consistently greater in the STR than in the original static suspension culture (Low RPM: 1.69-fold relative doubling, SD 0.42; Medium RPM: 2.51-fold relative doubling, SD 0.00; High RPM: 2.19-fold relative doubling, SD 0.18). Because spheres formed at low and medium RPM in the 0.25L STR and the relative doublings were twice that of the static suspension control, and because of the potential for scalability, the STR modality was selected for further optimization.

[0109] Mixing modeling research Next, experiments with 22 DASboxes were tried to evaluate different agitation strategies. Reactors operated at a single RPM over the duration of the culture did not allow sphere growth with limited attachment to the vessel walls. In contrast, when the RPM was graded over the course of the culture, we confirmed that small spheres were successfully formed at the beginning of the run and then grew without settling. Gradient agitation allowed sphere formation similar in appearance to spheres from our static culture modality (Figure 1 Panel H).

[0110] The data generated from these trials was used for CFD modeling of eight different agitation speeds. The hydrodynamic parameters were found to vary differently as a function of RPM (Figure 2 Panel A). At the same scale but different RPMs, the average energy dissipation (m 2 / s 3 ) and power per unit capacity (W / m 3 ) produced exponential curves, whereas maximum shear rate (1 / s), average shear rate (1 / s), volume average velocity (m / s) and tip speed (m / s) had linear slopes. The slopes were different in each case, indicating that the various hydrodynamic conditions do not scale equally based on RPM. By modeling aggrecan expression from 22 DASbox reactors with various hydrodynamic conditions, we found that maximum shear rate (1 / s) had the highest correlation with aggrecan expression (p=0.012) (Figure 2 Panel B).

[0111] CFD models of various agitation profiles showed which indicators influenced cell growth in the STR (Figure 3 Panel A). As previously mentioned, maximum shear rate (1 / s) influenced cell doubling (p=0.018) as well as aggrecan production (p=0.012) (Figure 3 Panel B). Cell settling was also influenced by cell volume fraction, which meant that cell spheres could settle out of solution as predicted by mean eddy current dissipation (p=0.024) (Figure 3 Panel C). Finally, sphere size was influenced by the use of dynamic agitation, with the use of gradient vs. static agitation schemes significantly correlating with sphere size (p=0.033) (Figure 3 Panel D). These findings identified new set points for the DASbox that we predict could generate disc-derived cells with properties comparable to cells grown in static culture.

[0112] Process Tunable Modeling The relationship between cell doubling or potency (as reflected in biomarker levels measured by various methods, e.g., via PCR) was modeled and established with respect to input factors including donor attributes, media attributes, and process parameters. These models determined that 5 of 11 measured input factors affect doubling and 11 of 11 factors affect PCR potency. Regression models and a series of two-factor contour profilers were used to define process parameters for two high-risk donor attributes and acceptable ranges for two high-risk process parameters (Figure 5). Analysis of each combination of highest and lowest donor attributes provided insight into the impact of donor variation. Applicants used these insights to develop methods, protocols, and systems to obtain cell populations with desired performance attributes from donors with a wide variety of initial attributes. In some embodiments, altering media and process parameters to obtain cells with specific (or a range of) performance attributes may be referred to as "tunable."

[0113] Figure 5 shows the results from this study. The shaded areas in the contour profiler indicate the failure conditions for each of the quality indicators. The unshaded range across all conditions indicates the acceptable operating range.

[0114] Cell doublings were recorded and found to be normally distributed using an Anderson-Darling goodness-of-fit test (p>0.079). Despite all STR runs being performed within the previously established ranges using one factor in a single experiment, our multivariate experimental conditions resulted in 44% of all STR runs having no cell doublings and 84% failing to meet the process doubling requirements. The failure of previous univariate analysis to capture 84% of failure conditions establishes the need for multivariate experiments when characterizing bioprocesses across multiple allogeneic cell lines. The initial agitation speed can be selected to both maintain cells in suspension and allow for the formation of cell spheres - i.e., to prevent or reduce the ability of the cells and spheres to settle out of solution while avoiding sufficient forces to break up or disrupt the cell spheres.

[0115] Comparison of static versus STR modalities An agitation gradient profile that demonstrated the ability to generate spheres with minimal settling or adhesion was developed and used for subsequent passaging stream growth between 0.25 L STR and static suspension culture (Figure 4A). In both modalities, all cells formed characteristic spheres (Figure 4B). Sphere size, aggrecan expression, and cell doublings did not exhibit statistically significant differences between the new STR process and the original static suspension method (Figure 4C). Flow cytometry showed no significant differences between the two modalities (Figure 4D).

[0116] Cells from both modalities were then tested in an in vivo rabbit model of intervertebral disc degeneration. After the initial injury, X-ray analysis showed that the disc height index was reduced by 25-50%. After dosing (Figure 5A), the disc height recovered slightly in the vehicle group and more substantially in the cell therapy group (p=0.019) (Figure 5B). Cells grown using either process had significantly higher disc heights than sham after 6 weeks in vivo (p>0.05).

[0117] Histological analysis by a pathologist identified abnormally high density and cellularity in the nucleus pulposus from sham or vehicle-treated discs, which was hypothesized to result from loss of hydration caused by the needle puncture injury and subsequent degeneration. In contrast, discs injected with cell therapy had a more normal appearance in these parameters (Figure 5C). Dorsal cartilage and osteophyte formation was noted, but was not associated with the treatment conditions and was attributable to the injury model. No inflammation or toxicity was noted that was likely caused by the cell therapy. Also, new vehicles (containing the original excipients, except one, at different concentrations) did not result in any abnormal findings. Histologically, no abnormal tissues (fat, bone, etc.) were noted in discs with cell therapy, which may explain the structural increase in disc height measured via x-ray.

[0118] Scaling up to large-scale STR The modeled cell volume fraction is expressed as a function of tip speed (m / s) or average energy dissipation (m 2 / s 3 showed that a scale-up strategy based on 1 / s shear can result in most cells settling out of solution. Also, scale-up using average shear (1 / s) can result in extremely high RPMs that can prevent sphere formation and promote cell death. 3), maximum shear rate (1 / s), and volume average velocity (m / s) based scale-up can all allow sphere formation while limiting cell settling. Of these hydrodynamic conditions, maximum shear rate (1 / s) had the strongest correlation with aggrecan expression and was therefore selected for scaling up to 50 L.

[0119] Cells were passaged and grown in 0.25L and 50L STR (Figure 6A). Maximum shear rate (1 / s) was maintained between scales. Spheres were successfully formed at both 0.25L and 50L scales (Figure 6B). 50L had comparable doublings (1.00 doublings compared to DASbox average) (Figure 6C). Sphere size was also comparable between the two DASboxes (relative sphere size 1.00, SD 0.07) and 50L (relative sphere size DASbox 0.98, SD 0.44). Finally, flow cytometry found no significant differences in surface markers (Figure 6D).

[0120] Analysis of intervertebral disc-derived cells When cells from five separate donors were grown in both static flask culture and STR, the resulting forward scatter histograms differed between methods. Notably, cells generated in STR were more uniform in size and smaller (Figure 11). Also, differences were observed in collagen 1 production between the two modalities, but not in total collagen production (Figure 12). Neither aggrecan nor sGAG showed differences between modalities (Figure 12). When other ECM molecules were evaluated, both lumican and collagen 6A2 showed significant expression and differences between modalities (Figure 12).

[0121] The cells not only produce extracellular matrix, but also have anti-inflammatory properties as measured via flow cytometry using the CSFE dye incorporation method (Figure 13). The proliferation of activated PBMCs (blood cells) is suppressed in the presence of disc-derived cells, indicating an immunomodulatory effect that may be beneficial in treating disc degeneration.

[0122] Although multiple embodiments have been disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description. As will become apparent, the present invention is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the present invention. Thus, the detailed description should be regarded as illustrative in nature and not restrictive.

[0123] All references disclosed herein, whether patent or non-patent, are hereby incorporated by reference as if each were incorporated by citation in its entirety. In the event of a conflict between a reference and this specification, the present specification, including definitions, will control.

[0124] Although the present disclosure has been described in some detail, it is understood that the disclosure is made by way of example and that changes in detail or structure can be made without departing from the spirit of the present disclosure as defined in the appended claims.

Claims

1. A method for culturing mammalian cells, comprising: collecting a plurality of mammalian cells; introducing the plurality of mammalian cells into a container or vessel containing a spheroid culture medium to produce a cell / media mixture; agitating the cell / media mixture at an agitation speed corresponding to a first energy level, wherein the first energy level is sufficient to maintain more than 90% of the cells in a suspended state; growing and dividing the plurality of cells to form cell spheroids having a first diameter; raising the agitation speed to a second energy level, wherein the second energy level is higher than the first energy level and is sufficient to maintain the cell spheroids in a suspended state; maintaining the agitation speed to allow the cell spheroids to achieve a larger diameter while maintaining a suspended state comprising the steps of.

2. The method according to claim 1, wherein the mammalian cells are selected from progenitor cells, stem cells, or pluripotent cells.

3. The method according to claim 1 or 2, wherein the mammalian cells are derived from muscle, brain, spinal cord, peripheral nerve, kidney, eye, skin, blood vessel, hair follicle, amnion, chorion, umbilical cord, placenta, liver, heart, lung, pancreas, cartilage, bone, thymus, thyroid, blood, lymph node, cartilage, and intervertebral disc cells.

4. The method according to claim 3, wherein the mammalian cells are chondrocytes.

5. The method according to claim 3, wherein the mammalian cells are intervertebral disc cells.

6. The method according to claim 3, wherein raising the agitation speed to the second energy level is linear over time.

7. The method according to claim 3, wherein increasing the stirring speed to a second energy level is non-linear. **Claim 8** The method according to claim 3, wherein increasing the stirring speed to a second energy level comprises at least one step. **Claim 9** The method according to claim 3, wherein the spheroid culture medium lacks scaffold molecules. **Claim 10** The method according to claim 3, wherein the plurality of mammalian cells are grown in adherent culture before being introduced into a container or vessel containing the spheroid culture medium. **Claim 11** The method according to claim 3, wherein the cells attached to the solid surface are doubled before the plurality of mammalian cells are introduced into a container or vessel containing the spheroid culture medium. **Claim 12** A method for culturing a mammalian cell population in a dynamic suspension state, comprising: On the first day, introducing a mammalian cell population into a bioreactor containing a spheroid culture medium to produce a cell / media mixture; Stirring the cell / media mixture at a first stirring speed; Causing cell spheroids to form in the cell population of the cell / media mixture; Stirring the cell / media mixture at a second speed, wherein the difference from the first speed results in a first shear value less than a first maximum shear value and the second speed results in a second shear value less than a second maximum shear value. A method comprising the above steps. **Claim 13** Introducing a mammalian cell population into a bioreactor containing a spheroid culture medium to produce a cell / media mixture; Stirring the cell / media mixture at a first stirring speed sufficient to prevent or arrest loss of the suspension state of the cells; Causing cell spheroids to form in the cell population in the cell / media mixture. Maintaining most cells in a suspended state, Isolating and recovering the suspended cell spheres, whereby Dynamically culturing mammalian cell spheres, A method for the dynamic culture of mammalian cell spheres.

14. A method for modifying one or more characteristics of a therapeutic mammalian cell population, comprising: Isolating a population of cells from a donor tissue, wherein the donor has a first attribute with a first attribute score and a second attribute with a second attribute score; Determining the characteristics desired in the therapeutic cell population; Selecting a first medium parameter based on the first attribute score and / or the second attribute score; Selecting a process parameter based on the first attribute score and / or the second attribute score and / or the medium parameter; Culturing the population of cells in suspension in a container or vessel containing a spheroid culture medium; Maintaining the population of cells in suspension; Dividing and growing the cells to form clonal cell spheres; Maintaining the population of cell spheres in suspension; Isolating and recovering the suspended cell spheres having the predetermined characteristics, whereby Modifying one or more characteristics of a therapeutic mammalian cell population A method comprising the above steps.

15. The method according to any one of claims 12 to 14, wherein the mammalian cells are selected from progenitor cells, stem cells, or pluripotent cells.

16. The method according to claim 15, wherein the mammalian cells are derived from muscle, liver, heart, lung, pancreas, bone, thyroid, blood, lymph node, brain, spinal cord, peripheral nerve, kidney, eye, skin, blood vessel, hair follicle, amnion, chorion, cartilage, intervertebral disc cells, umbilical cord, and placenta.

17. The method according to claim 15, wherein the mammalian cells are chondrocytes.

18. The method according to claim 15, wherein the mammalian cells are intervertebral disc cells.

19. The method according to claim 15, wherein increasing the stirring speed to the second energy level is linear over time.

20. The method according to claim 15, wherein increasing the stirring speed to the second energy level is non-linear.

21. The method according to claim 15, wherein increasing the stirring speed to the second energy level comprises at least one step.

22. The method according to claim 15, wherein the spheroid culture medium lacks a scaffold molecule.

23. The method according to claim 15, wherein the plurality of mammalian cells are grown in adherent culture before being introduced into a container or vessel containing the spheroid culture medium.

24. The method according to claim 15, wherein the cells attached to the solid surface are doubled before the plurality of mammalian cells are introduced into a container or vessel containing the spheroid culture medium.

25. More than 90% of the cells are negative for surface markers selected from CD24, HLA-DR / DP / DQ, CD45, CD40, CD271, CD80, CD86, or a combination thereof, positive for surface markers selected from CD44, CD73, CD90, HLA-ABC, or a combination thereof, intervertebral disc cells A mammalian cell population comprising

26. More than 90% of the cells Express one or more of aggrecan, collagen 1, collagen 2, collagen 6, collagen 14, decorin (DCN), biglycan (BGN), lumican (LUM), and fibromodulin (FMOD); exhibit one or more of the anti-inflammatory effects confirmed by an activated T cell assay, Intervertebral disc cells A mammalian cell population comprising

27. A method for culturing mammalian cells, comprising Collecting a plurality of mammalian cells, Introducing the plurality of mammalian cells into a container or vessel containing a sphere culture medium to produce a cell / media mixture, Stirring the cell / media mixture at a stirring speed corresponding to a first energy level, wherein the first energy level is sufficient to maintain more than 90% of the cells in a suspended state, Growing and dividing the plurality of cells to form cell spheres of a first diameter, Maintaining the stirring speed to allow the cell spheres to achieve a larger diameter while maintaining a suspended state A method comprising

28. The method according to claim 27, wherein the mammalian cells are selected from progenitor cells, stem cells, or pluripotent cells.

29. The method according to claim 27 or 28, wherein the mammalian cells are derived from muscle, brain, spinal cord, peripheral nerve, kidney, eye, skin, blood vessel, hair follicle, amnion, chorion, umbilical cord, placenta, liver, heart, lung, pancreas, cartilage, bone, thymus, thyroid, blood, lymph node, cartilage, and intervertebral disc cells.

30. The method according to claim 29, wherein the mammalian cells are chondrocytes.

31. The method according to claim 29, wherein the mammalian cell is an intervertebral disc cell.

32. The method according to claim 29, wherein the spheroid culture medium lacks a scaffold molecule.

33. The method according to claim 29, wherein the plurality of mammalian cells are grown in adherent culture before being introduced into a container or vessel containing the spheroid culture medium.

34. The method according to claim 29, wherein the number of cells attached to a solid surface is doubled before the plurality of mammalian cells are introduced into a container or vessel containing the spheroid culture medium.

35. The method according to claim 29, comprising increasing the stirring speed to a second energy level, wherein the second energy level is higher than the first energy level and is sufficient to maintain the cell spheroids in a suspended state.

36. The method according to claim 35, wherein increasing the stirring speed to the second energy level is linear over time.

37. The method according to claim 35, wherein increasing the stirring speed to the second energy level is non-linear.

38. The method according to claim 37, wherein increasing the stirring speed to the second energy level comprises at least one step.

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