Manufacturing methods for cell therapies

Abstract

The present invention relates to a method for expansion of adherent cells, including mesenchymal stem cells (MSCs) such as adipose-derived stromal stem cells (ASCs), in microcarrier-based bioreactor systems using different culture medium formulations.

Description

[0001] Manufacturing methods for cell therapies

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a method for expansion of adherent cells, including mesenchymal stem cells (MSCs) such as adipose-derived stromal stem cells (ASCs), in microcarrier-based bioreactor systems using different culture medium formulations.

[0004] BACKGROUND OF THE INVENTION

[0005] In the developing medical world, a growing need exists for cells in large amounts for their use in research and medical applications. In particular, adult stem cell therapy is continuously developing for treating and curing various conditions such as fistulas, leukemia, lymphoma, neurodegenerative diseases, brain and spinal cord injury, heart diseases, blindness and vision impairment, pancreatic beta cell loss of function, cartilage repair, osteoarthritis, musculoskeletal diseases, wounds, infertility, autoimmune diseases and inflammatory diseases such as inflammatory bowel disease and Crohn’s Disease. An overview of current medical applications for the different types of stem cells may be found in Hoang, DM, Signal Transduction and Targeted Therapy 7, 272, (2022).

[0006] Stem cells for use in research and medical applications may be derived from embryonal, fetal or adult tissue and include embryonic stem cells (ESCs), umbilical cord stem cells, induced pluripotent stem cells (iPSCs) and adult stem cells from different sources. Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into a variety of cell types including osteoblasts, chondrocytes, myocytes and adipocytes.

[0007] Darvadstrocel (INN) is the first commercial allogeneic mesenchymal stem cell therapy to receive central marketing authorization approval in Europe and is currently approved for the treatment of refractory complex perianal fistulas (CPAF) in patients suffering from Crohn’s Disease.

[0008] The manufacturing process for Darvadstrocel starts from a lipoaspirate of adipose tissue, from which adipose stem cells (ASCs) are extracted and expanded in static 2D-systems to generate the final drug substance (FDS).

[0009] Since the culturing in static 2D-systems has only limited process scalability, there is a need for novel methods of cell expansion which allow to obtain greater numbers of stem cells which can be processed to the final drug substance.

[0010] There have been approaches for cell expansion by 3D culture, e.g. in WO 2007 / 108003 A2, describing a method comprising culturing adherent cells from placental or adipose tissue under three-dimensional culturing conditions. In another approach, WO 2021 / 250583 A2 relates to methods of serum-free stem cell culture, describing 3D cultures in bioreactors as well as cell culture media and compositions for use in the same. Further, WO 2021 / 009777 A2 describes methods of culturing stem cells using three-dimensional methods, wherein said method is either a spheroid-based method or a microcarrier-based method, leading to an expanded population of the stem cells, and a stem cell derived-conditioned medium obtained from this process. WO 2021 / 108243 A1 describes methods for expansion of human pluripotent stem cells in a bioreactor using xeno-free media and discloses further steps of cell harvest, concentration and cryopreservation. In another approach, WO 2015 / 131143 A1 relates to methods for expansion of stem cells, such as mesenchymal stem cells, and their further application in the treatment of conditions such as ischemic cardiovascular and cerebral injuries. In another approach, Cunha et al. (J Biotechnol. (2015) 213: 97-108) describe a continuous perfusion culture of human mesenchymal stem cells in bioreactors and the further steps of washing and concentrating of the cells using a tangential flow filtration system. In another approach, Cunha et al. (J Biotechnol. (2017) 248: 87-98) describe a method for scale-up of mesenchymal stem cell expansion and harvesting from spinner flasks to bioreactors. In yet another approach, Sousa et al. (Biotechnol Prog. (2015) 31 (6) :1600-12) describe the impact of different bioreactor designs for the scale-up of microcarrier-based stem cell cultures.

[0011] Nevertheless, there is still a need for scalable and more efficient expansion processes for adherent cells, in particular adipose tissue-derived stem cells, leading to higher cell yields.

[0012] SUMMARY OF THE INVENTION

[0013] The present invention is directed to a method for expansion of adherent cells, including mesenchymal stem cells (MSCs) such as adipose-derived stromal stem cells (ASCs), using different culture medium formulations. The method comprises the steps of thawing said adherent cells, directly inoculating said thawed adherent cells in a cell culture medium comprising microcarriers such that the adherent cells attach to the microcarriers, and culturing said adherent cells on said microcarriers.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Colonization of ASCs on Plastic Plus, Corning Enhanced Attachment (CEA) or LC Synthemax II microcarriers over culture time in a 0.2 L stirred-tank DASbox bioreactor using DMAX with 10% FBS (A) and colonization of ASCs on Plastic Plus or LC Synthemax II microcarriers over culture time in a 0.2 L stirred-tank DASbox bioreactor using RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC- CC (B). Data in (B) (n=3; 2 biological replicates) are represented as mean ± SD. p < 0.01 (**), p < 0.001 (***), p < 0.0001 (*"*).

[0016] Figure 2: Colonization of ASCs on 16 g / L or 22 g / L of LC Synthemax II microcarriers over culture time in a 2 L stirred-tank bioreactor in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC- CC.

[0017] Figure 3: Cell concentration and viability (A) and respective cumulative population doublings (PDs) (B) throughout ASC expansion in a 2 L stirred-tank bioreactor containing LC Synthemax II microcarriers (16 g / L or 22 g / L) in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC.

[0018] Figure 4: Cell concentration and viability throughout ASC expansion in a 2 L stirred-tank bioreactor containing 22 g / L of LC Synthemax II microcarriers using DMAX with 10 % FBS.

[0019] Figure 5: Microcarrier colonization (A) and viable cell growth profile (B) of ASCs transferred to fresh microcarriers by single cell inoculation or bead-to-bead transfer at a populated:empty microcarrier ratio of 1 :4 or 1 :9.

[0020] Figure 6: Cell characteristics of ASCs after using single cell re-inoculation or bead-to-bead transfer at a populated:empty microcarrier ratio of 1 :4 or 1 :9 as scale-up strategies. (A) Cumulative PDs of ASC expanded in the stirred-tank bioreactor. (B) Percentage of cell recovery after microcarrier / cell separation and cell centrifugation and (C) cell viability after dissociation with TrypLE Select reagent. (D) Flow cytometry analysis of the MSC characteristic surface markers (CD29, CD90, CD73 and CD105) after harvest and cryopreservation.

[0021] Figure 7: Comparison of overall cell recovery yields using counterflow centrifugation or standard centrifugation to concentrate cells prior to their cryopreservation (n=5; p < 0.05 (*)).

[0022] Figure 8: Comparison of cell characteristics of ASCs after applying different methods of cell concentration. (A) Viability, (B) expression of characteristic surface markers (CD29 / CD90 and CD73 / CD105) and (C) diameter of ASC harvested after culture in stirred-tank bioreactors (STB) and concentrated by standard centrifugation procedures or using counterflow centrifugation. ASC cultured under static conditions were harvested using TrypLE™ Select dissociation agent and concentrated by standard centrifugation. (D) Concentration of kynurenine after 24h induction with IFN-y, obtained for ASC cultured in microcarrier-supported stirred-tank bioreactor cultures. (E) % of Inhibition of lymphoproliferation by ASC expanded in static or STB, concentrated by standard centrifugation or counterflow centrifugation. Data (n=5) are represented as mean ± SD. p < 0.05 (*).

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] Where the term “comprise” or “comprising” is used in the present description and claims, it does not exclude other elements or steps. For the purpose of the present invention, the term “consisting of” is considered to be an optional embodiment of the term “comprising of". If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which optionally consists only of these embodiments.

[0025] Where an indefinite or a definite article is used when referring to a singular noun e.g. “a” or “an”, “the”, this includes a plural form of that noun unless specifically stated. Vice versa, when the plural form of a noun is used it refers also to the singular form.

[0026] Furthermore, the terms first, second, third or (a), (b), (c) and the like in the description and in the claims are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0027] In the context of the present invention any numerical value indicated is typically associated with an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. As used herein, the deviation from the indicated numerical value is in the range of ± 10%, and preferably of ± 5%. The aforementioned deviation from the indicated numerical interval of ± 10%, and preferably of ± 5% is also indicated by the terms “about” and “approximately” used herein with respect to a numerical value.

[0028] Adherent cells include, but are not limited to, fibroblasts, cardiomyocytes, osteoblasts, adipocytes, chondrocytes, endothelial cells, retinal pigment epithelial (RPE) cells, dendritic cells and stem cells.

[0029] In a preferred embodiment, the adherent cells are stem cells. The stem cells may be pluripotent stem cells, neural stem cells or mesenchymal stem cells (also referred to herein as “MSCs”).

[0030] There are two sources of pluripotent stem cells. First, embryonic stem cells (ESCs) are derived from the inner cell mass of a pre-implantation blastocyst and pluripotency is controlled by an intrinsic regulatory network of core transcription factors, octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2), and Nanog homeobox (NANOG). In one embodiment, an embryonic stem cell line is used. An embryonic stem cell line comprises constantly dividing cells produced from a group of parent cells which were harvested from a single embryo. The embryonic stem cell line used in the present invention is not obtained by destruction of a human embryo. Embryonic stem cell lines are commercially available, for example from ATCC. The embryonic stem cells of the embryonic stem cell line do not lose their pluripotency while they are in culture. In particular, the embryonic stem cells of the embryonic stem cell line do not differentiate while they are in culture. Second, induced pluripotent stem cells (IPSCs) are derived by the ectopic or elevated expression of four transcription factors, OCT4, SOX2, Kruppel like factor 4 (KLF4), and MYO proto-oncogene (C-MYC) essential for induction of pluripotency in somatic cells.

[0031] Techniques for isolating stable (undifferentiated) cultures of embryonic stem cells, such as human embryonic stem cells, are well established (e.g. US 5,843,780; Thomson et al., Science (1998) 282: 1145-1147; Turksen & Troy (2006) Human Embryonic Stem Cells. In: Turksen K. (eds) Human Embryonic Stem Cell Protocols. Methods in Molecular Biology, volume 331 , Humana Press; Sevilla et al., Stem Cell Research (2017) 25: 217 220; and Mitalipova & Palmarini (2006) Isolation and Characterization of Human Embryonic Stem Cells. In: Turksen K. (eds) Human Embryonic Stem Cell Protocols. Methods in Molecular Biology, volume 331 , Humana Press). In one embodiment, the method for obtaining embryonic stem cells does not include the destruction of one or more human embryos.

[0032] Techniques for producing induced pluripotent stem cells (iPSCs) are well established since their discovery in 2007 by Yamanaka's group (e.g. Takahashi et al., Cell (2007) 131 (5): 861 -72). Since then, new improved methods for IPSC generation have been developed, including non-integration and feeder free methodologies and automated high-throughput derivation (Pauli et al., Nature Methods (2015) 12(9): 885-892).

[0033] IPSCs are characterized by the expression of a battery of pluripotency markers: NANOG, SOX2, SSEA4, TRA1 - 81 , TRA1-60, and the lack of lineage-specific markers. The pluripotency of iPSC is demonstrated by their capacity to differentiate into the three germ layers in the embryoid body assay, with posterior analysis of differentiation markers from the three germ layers Tuj1 (ectoderm marker), SMA (mesoderm marker) and SOX17 (endoderm marker) by immunohistochemistry (Pauli et al., Nature Methods (2015) 12(9): 885-892).

[0034] Neural stem cells are derived from embryonic neural tissue and can self-renew and further differentiate into committed neural sub-types, such as neurons, astrocytes, or oligodendrocytes. Neural stem cells are multipotent, meaning they have a more limited range of differentiation capability compared to pluripotent stem cells, but they are still capable of forming all cell types in the central nervous system. Said neural stem cells are primarily found in specific areas of the brain, such as the subventricular zone and the subgranular zone of the hippocampus, where they contribute to neurogenesis (the formation of new neurons) throughout life, although at reduced rates in adults. Key markers for identifying and characterizing neural stem cells include, but are not limited to, Nestin, Sox2, Pax6, BLBP (Brain Lipid-Binding Protein) and Mashl (Zhang et al., Biomed Res Int 2015:727542).

[0035] Preferably, the stem cells are MSCs, most preferably the stem cells are adipose-derived stromal stem cells (also referred to herein as “ASCs”).

[0036] Mesenchymal stem cells (MSCs) are multipotent stromal cells which are typically derived from connective tissue and are non-hematopoietic cells. According to Dominici et al. (2006), Cytotherapy 8(4): 315-317 MSCs are characterized in that they: (1 ) adhere to plastic under standard culture conditions (e.g. a minimal essential medium plus 20% fetal bovine serum (FBS)); (2) express (i.e. greater than or equal to 80% of a population of MSCs) CD105, CD90, CD73 and CD44; (3) lack expression (e.g. less than or equal to 5% of the MSC population express) of CD45, CD14 or CD11b, CD790L or CD19, and HLA-DR (HLA Class II); (4) are capable of differentiating into osteoblasts, adipocytes and chondroblasts. MSCs can be obtained using standard methods from, for example, bone marrow, umbilical cord tissue and blood, menstrual, dental pulp, cord blood, placental and adipose tissues. Although MSCs obtained from different tissues are similar, they have some differences in phenotypical and functional characteristics. For example, the expression levels of cell surface markers CD54 and CD106 may differ depending on the source / origin of the MSCs. These expression levels can be measured by flow cytometry. The mRNA levels of some genes such as SOX2, IL1 a, IL1 p , IL6 and ILS, may be differentially expressed by MSCs from different tissues, and can be measured by routine methods. IL6 and PGE2 secretion may also be different between MSCs from different origins, and thus the cells may have different modulatory capacity (see, e.g. Yang et al. PLoS ONE (2013) 8(3) e59354). Preferably, the MSCs are derived from adipose tissue, i.e. are adipose tissue-derived stem cells.

[0037] Bone marrow-derived MSCs (BM-MSCs) are similar to MSCs from other tissue sources. However, they show some differences in phenotypical and functional characteristics compared to MSCs from other tissue origins, such as umbilical-cord MSCs, placental MSCs, dental pulp MSCs, and menstrual MSCs. Even though their minimal characterization criteria are common, including their capacity to adhere to plastic, minimal surface identity markers and capacity to differentiate into bone, cartilage, tendon and fatty tissue, they all have some slight differences. These peculiarities include different expression levels of some surface markers, such as CD105, different levels of secreted soluble factors implicated in their immunomodulatory potential and regenerative potential, and in general, slightly different functional properties that may make each source or origin more suitable for specific therapeutic indications (Miura et al., Int J Hematology (2016) 103(2): 122-128; Wuchter et al., Cytotherapy (2015) 17(2): 128- 139; Wright et al., Stem Cells (2011 ) 29(2): 169-178).

[0038] Huang et al. (J. Dent. Res. (2009) 88(9): 792-806) discusses MSCs from dental pulp and compares their characteristics with MSCs from other sources. Carvalho et al. (Curr Stem Cell Res Then (2011 ) 6(3): 221 -228) and Harris et al. (Curr Stem Cell Res Then (2013) 8(5): 394-399) discuss umbilical cord-derived MSCs, their characterization (including phenotype and secretome) and applications thereof.

[0039] Adipose tissue-derived MSCs (ASCs) are normally isolated from subcutaneous adipose tissue, which allows them to be acquired in large numbers. ASCs proliferate rapidly with a high cellular activity, making them an ideal source for obtaining MSCs.

[0040] By “adipose tissue” is meant any fat tissue. The adipose tissue may be brown or white adipose tissue, derived from subcutaneous, omental / visceral, mammary, gonadal, or other adipose tissue site. Typically, the adipose tissue is subcutaneous white adipose tissue. The ASCs may comprise a primary cell culture or an immortalized cell line. Preferably, the ASCs are a primary cell culture. Also preferably, the ASCs have not been genetically modified. The adipose tissue may be from any organism having fat tissue. Typically, the adipose tissue is mammalian adipose tissue, most typically the adipose tissue is human adipose tissue. A convenient source of adipose tissue is from liposuction surgery, however, the source of adipose tissue or the method of isolation of adipose tissue is not critical to the invention.

[0041] Preferred ASCs are the human expanded allogeneic adipose-derived stem cells (human eASCs) as provided in the product Darvadstrocel. These expanded ASCs express the cell surface markers CD29, CD73, CD90 and CD105. The cells are capable of expressing factors such as vascular endothelial growth factor (VEGF), transforming growth factor-beta 1 (TGF-|31 ), interleukin 6 (IL-6), matrix metalloproteinase inhibitor-l (TIMP-1 ) and interferon-gamma (IFN-y) and inducible indoleamine 2,3-dioxygenase (IDO). Thus, the population of ASCs may be characterized in that at least about 50%, at least about 60%; at least about 70%; at least about 80%; at least about 85%; at least about 90% or at least about 95% or more express one or more of CD29, CD73, CD90 and / or CD105. The population of ASCs may be characterized in that at least about 50%, at least about 60%; at least about 70%; at least about 80%; at least about 85%; at least about 90% or at least about 95% of the population of cells express all of CD29, CD73, CD90 and CD105. Typically, the population of ASCs may be characterized in that at least about 80% of the population of cells express all of CD29, CD73, CD90 and CD105.

[0042] According to Bourin et al. (Cytotherapy (2013) 15(6): 641 -648), a population of ASCs may be defined as being positive for expression of CD13, CD29, CD44, CD73, CD90 and CD105, and negative for expression of CD31 and CD45. In the population of ASCs, at least about 50%, at least about 60%; at least about 70%; at least about 80%; at least about 85%; at least about 90% or at least about 95% of the population of cells may express CD13, CD29, CD44, CD73, CD90 and CD105, and fewer than about 5%, about 4%, about 3% or about 2% of the population of ASCs may express CD31 and CD45. Typically, in the population of ASCs, at least about 80% of the population of cells may express CD13, CD29, CD44, CD73, CD90 and CD105, and fewer than about 5% of the population of ASCs may express CD31 and CD45.

[0043] The ASCs may be adherent to plastic under standard culture conditions. Expanded ASCs (eASC) have been cultured in vitro for one or more generations to increase the number of ASCs. The expanded ASCs retain at least one and preferably all biological functions of the ASC, such as the ability to adhere to plastic and the ability to differentiate into one or more cell types. Typically, eASCs exhibit a fibroblast-like morphology in culture. Specifically, these cells are big and are morphologically characterized by a shallow cell body with few cell projections that are long and thin. The nucleus is large and round with a prominent nucleolus, giving the nucleus a clear appearance. Most of eASCs display this spindle-shaped morphology, but it is usual that some of the cells acquire polygonal morphologies (Zuk et al. Tissue Eng (2001 ) 7(2): 211-228).

[0044] The ASCs may be positive for the surface markers HLA I, CD29, CD44, CD59, CD73, CD90, and CD105. In some embodiments, the population of ASCs may be characterized in that at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%; at least about 90% or at least about 95% of the population of ASCs express the surface markers HLA-I, CD29, CD44, CD59, CD73, CD90, and CD105. Typically, at least about 80% of the eASCs express the surface markers HLA I, CD29, CD44, CD59, CD73, CD90, and CD105.

[0045] The ASCs may be negative for the surface markers HLA II, CD11 b, CD11 c, CD14, CD45, CD31 , CD80 and CD86. In some embodiments, the population of ASCs may be characterized in that fewer than about 5% of the population of ASCs express the surface markers HLA II, CD11 b, CD11 c, CD14, CD45, CD31 , CD80 and CD86. More typically, fewer than about 4%, 3% or 2% of the population of ASCs express the surface markers HLA II, CD11 b, CD11c, CD14, CD45, CD31 , CD80 and CD86. In one embodiment, fewer than about 1 % of the population of ASCs express the surface markers HLA II, CD11 b, CD11 c, CD14, CD45, CD31 , CD80 and CD86.

[0046] In some cases, in a population of ASCs at least about 80% of the population of cells express all of CD29, CD73, CD90 and CD105 and fewer than about 5% of the population of ASCs express the surface markers HLA II, CD11 b, CD11c, CD14, CD45, CD31 , CD80 and CD86.

[0047] In some embodiments the population of ASCs may express one or more (e.g. two or more, three or more, four or more, five or more, six or seven) of HLA I, CD29, CD44, CD59, CD73, CD90, and CD105. In some embodiments, the eASCs may not express one or more (e.g. two or more, three or more, four or more, five or more, six or more, seven or eight) of HLA II, CD11 b, CD11 c, CD14, CD45, CD31 , CD80. In some embodiments, the eASCs express four or more of HLA I, CD29, CD44, CD59, CD73, CD90, and CD105 and do not express four or more of HLA II, CD11 b, CD11c, CD14, CD45, CD31 , CD80.

[0048] Expression of CD34 may be negative or low, e.g. expressed by 0 to about 30% of the population of ASCs. Thus, in some cases, the ASCs as described above may express CD34 at low levels, e.g. in about 5 to about 30% of the population. Alternatively, in other cases, the ASCs as described do not express CD34, e.g. fewer than about 5% of the population of ASCs express CD34.

[0049] In some embodiments, the population of ASCs (e.g. at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%; at least about 90% or at least about 95% of the population of cells) may express one or more (e.g. two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more (e.g. up to 13)) of the markers CD9, CD10, CD13, CD29, CD44, CD49A, CD51 , CD54, CD55, CD58, CD59, CD90 and CD105. For example, the ASCs may express one or more (e.g. two, three or all) of the markers CD29, CD59, CD90 and CD105, e.g. CD59 and / or CD90.

[0050] In some embodiments the population of ASCs may not express one or more (e.g. two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more (e.g. up to 15)) of the markers Factor VIII, a-actin, desmin, S-100, keratin, CD11 b, CD11c, CD14, CD45, HLA II, CD31 , CD45, STRO-I and CD133, e.g. the ASCs do not express one or more (e.g. two, three or all) of the markers CD45, CD31 and CD14, e.g. CD31 and / or CD45.

[0051] In certain embodiments, the ASCs as described above (I) do not express markers specific for antigen presenting cells (APCs); (II) do not express IDO constitutively; and / or (ill) do not significantly express MHC II constitutively. Typically, expression of IDO or MHC II may be induced by stimulation with IFN-y. In certain embodiments, the ASCs as described above do not express Oct4.

[0052] Methods for the isolation and culture of ASCs to provide eASCs and populations of stem cells, and compositions comprising stem cell populations are known in the art. ASCs are typically prepared from the stromal fraction of adipose tissue and are selected by adherence to a suitable surface e.g. plastic. Thus, the methods of culturing stem cells disclosed herein may comprise an initial step (prior to step (a) of any one of the methods) of: (I) isolating a population of ASCs from the stromal fraction of adipose tissue obtained from a patient, and (ii) culturing the population of ASCs to obtain expanded ASCs (eASCs). The ASCs can optionally be selected in step (i) for adherence to a suitable surface, e.g. plastic. Optionally, the phenotype of the ASCs may be assessed during and / or subsequent to the culturing step (ii). ASCs can be obtained by any standard means in the art. Typically, said cells are obtained disassociating the cells from the source tissue (e.g. lipoaspirate or adipose tissue), typically by treating the tissue with a digestive enzyme such as collagenase. The digested tissue matter is then typically filtered through a filter of between about 20 microns to 1 mm. The cells are then isolated (typically by centrifugation) and cultured on an adherent surface (typically tissue culture plates or flasks). Such methods are known in the art and e.g. as disclosed in US. Patent No. 6,777,231 .

[0053] According to this methodology, lipoaspirates are obtained from adipose tissue and the cells are derived therefrom. In the course of this methodology, the cells may be washed to remove contaminating debris and red blood cells, preferably with PBS. The cells are digested with collagenase (e.g. at 37°C for 30 minutes, 0.075% collagenase; Type I, Invitrogen, Carlsbad, CA) in PBS. To eliminate remaining red blood cells, the digested sample can be washed (e.g. with 10% FBS), treated with 160 mmol / L NH4CI, and finally suspended in Dulbecco’s modified Eagle's (DMEM) complete medium (DMEM containing 10% FBS, 2 mM glutamine and 1% penicillin / streptomycin). The cells can be filtered through a 40 pm nylon mesh.

[0054] Cultured human ASCs are described in DelaRosa et al. (Tissue Eng Part A. (2009) 15(10): 2795-806) and Lopez- Santalla et al. (Stem cells (2015) 33: 3493-3503). In one embodiment (as described in Lopez-Santalla et al. (2015), cited above), human adipose tissue aspirates from healthy donors were washed twice with phosphate-buffered saline and digested with 0.075% collagenase (Type I; Invitrogen). The digested sample was washed with 10% FBS, treated with 160 mM NH4CI to eliminate the remaining erythrocytes, and suspended in culture medium (DMEM) with 10% FBS). Cells were seeded (2-3 x 104cells / cm2) in tissue culture flasks and cultured (37°C, 5% CO2) with change of culture medium every 3 to 4 days. Cells were transferred to a new flask (103cells / cm2) when they reached 90% confluence. Cells were expanded up to duplication 12 to 14 and frozen.

[0055] In another embodiment (as described by DelaRosa et al. (2009), Tissue Eng Part A 15(10): 2795-806), lipoaspirates obtained from human adipose tissue from healthy adult donors were washed twice with PBS and digested at 37°C for 30 minutes with 18 U / mL of collagenase type I in PBS. One unit of collagenase liberates 1 mM of L-leucine equivalents from collagen in 5 hours at 37°C, pH 7.5 (Invitrogen, Carlsbad, CA). The digested sample was washed with 10% of FBS, treated with 160 mM NH4CI, suspended in culture medium (DMEM containing 10% FBS), and filtered through a 40 pm nylon mesh. Cells were seeded (2-3 x 104cells / cm2) onto tissue culture flasks and expanded at 37°C and 5% CO2, changing the culture medium every 7 days. Cells were passed to a new culture flask when cultures reached 90% of confluence. Cells were phenotypically characterized by their capacity to differentiate into chondro-, osteo-, and adipogenic lineages.

[0056] The adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, are cultured in a suitable tissue culture vessel comprising a surface suitable for the adherence of cells, e.g. plastic. Non-adherent cells are removed, e.g. by washing with a suitable buffer, to provide an isolated population of adherent cells (e.g. ASC). Cells isolated in this way can be seeded (preferably at a density of 2-3 x 104cells / cm2) onto tissue culture flasks and expanded at 37°C and 5% CO2, changing the culture medium every 3 to 4 days. Cells are preferably detached from the adherent surface (e.g. by means of trypsin) and passed (“passaged”) to a new culture flask (preferably at a density of 1 ,000 cells / cm2) when cultures reach around 90% of confluence.

[0057] The adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, may be cultured for at least about 15, at least about 20 days, at least about 25 days, or at least about 30 days. Typically, the expansion of cells in culture improves the homogeneity of the cell phenotype in the population, such that a substantially pure population is obtained.

[0058] In some embodiments, the adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, are expanded in culture for at least three culture passages or “passaged at least three times". In other embodiments, the adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs are passaged at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times. It is preferable that cells are passaged more than three times to improve the homogeneity of the cell phenotype in the cell population. Indeed, the cells may be expanded in culture indefinitely so long as the homogeneity of the cell phenotype is improved, and the capacity for differentiation is maintained.

[0059] In some embodiments, the adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs are expanded in 3D culture for one to seven population doublings (PDs). Isolation of adherent cells, preferably stem cells, preferably MSCs and most preferably ASCs, is preferably carried out under sterile or GMP conditions.

[0060] After the initial step of (I) isolating a population of adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, from an organic source, e.g. adipose tissue, bone explants, myocardial tissue, skin tissue, cartilage, eyes, vessels, blood samples, skeletal muscle or other tissues, and the following step (II) of culturing the population of cells, said population of cells may be cryopreserved, preferably by using dimethyl sulfoxide (DMSO), more preferred by using 5% to 10% DMSO.

[0061] In a preferred embodiment, the cells are ASCs and after the initial step of (i) isolating a population of ASCs from the stromal fraction of adipose tissue obtained from a patient and the following step (ii) of culturing the population of ASCs as described in the paragraphs above, said population of ASCs may be cryopreserved, preferably by using DMSO, more preferred by using 5% to 10% DMSO.

[0062] In the next step, samples of said cryopreserved adherent cells, preferably stem cells, preferably MSCs and most preferably ASCs are thawed to be used in the method of the present invention.

[0063] The term thawing describes the process of bringing frozen or cryopreserved cells back to their functional, viable state at a controlled rate after storage at low temperatures. In one embodiment, thawing describes the process of rapidly transferring vessels containing the cryopreserved adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, to a prewarmed water bath having a temperature between 30°C and 40°C, preferably having a temperature of 37°C, until the content of the vessel is liquid, and diluting the thawed cells slowly in pre-warmed culture medium before further use, i.e. before directly inoculating the thawed cells in a cell culture medium comprising microcarriers. In another embodiment, thawing describes the process of transferring vessels containing the cryopreserved adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, to a dry thawing system such as the ThawSTAR Automated Cell Thawing System (STEMCELL Technologies), using thermal sensors to detect the temperature of the frozen vessels and automatically adjusting the heat applied to the vessels based on the starting temperature. The thawing time using a dry thawing system can be about 1 to 4 minutes, preferably about 2 to 3 minutes from the time of vessel insertion, followed by diluting thawed cells slowly in pre-warmed culture medium before further use, i.e. before directly inoculating the thawed cells in a cell culture medium comprising microcarriers.

[0064] Directly inoculating as used in the context of the present invention means that no seed train is applied after thawing the cryopreserved adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs. The term directly inoculating describes the process of immediately introducing said thawed cells after the thawing step into the culture medium comprising microcarriers without applying any intermediate culturing steps. This means that the thawed cells are introduced in the small volume obtained after the thawing step. The term seed train describes the process of scaling a small volume of cell culture to a larger volume to obtain a higher number of viable cells to inoculate larger cultivation systems. In a traditional seed train, this is achieved by intermediate steps of passaging cells from a working cell bank vial through increasingly larger cultivation systems including T-flasks, roller bottles or shake flasks, small scale bioreactor systems and subsequently larger bioreactors.

[0065] Culturing of adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, as used herein means that said cells are contained in a cell culture medium under conditions suitable for maintaining viability and supporting growth of said cells. Said conditions include maintaining cells at a temperature of 36 to 37°C and a pH of 7.4, optionally adding exogenous CO? at a concentration of 5% to 7%.

[0066] Cell culture systems can differ in the way required media components are added to the cell culture. The term batch cell culture refers to a system, wherein all the necessary components for the cell culture are added at the beginning of the process and culturing is allowed to proceed without any further addition of single compounds or cell culture medium. The term fed-batch cell culture refers to a system, wherein the initial components are added at the beginning of the process and wherein during the process, single compounds or cell culture medium are intermittently or continuously added to the cell culture without removing or replacing the culture medium. The term perfusion cell culture refers to a process, wherein fresh cell culture medium is supplied to the culture during the culturing and spent medium containing waste products is removed, preferably continuously. In one embodiment, the method of culturing adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs according to the present invention can be performed in the batch-, fed-batch-, or perfusion mode.

[0067] The skilled person will be aware of suitable cell culture media for supporting the growth of adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs. The terms "medium", "cell culture medium" and "culture medium" are used interchangeably herein. Cell culture media can be in liquid or solid form, including gelatinous media such as agar, agarose, gelatin and collagen matrices. Preferably, the cell culture medium is in liquid form. Most media generally are composed of four basic chemical groups: amino acids, carbohydrates, inorganic salts, and vitamins. This serves as the basis for a more complex medium, to which supplements such as serum, buffers, growth factors, lipids, and the like are added. Antibiotics such as gentamicin, penicillin and streptomycin can also be included in the cell culture medium to suppress the growth of bacteria in the culture. The concentration of gentamicin in the culture medium may be about 10 pg / ml to 100 pg / ml. The concentration of penicillin in the culture medium may be about 10 to 200 units per ml. The concentration of streptomycin in the culture medium may be about 10 pg / ml to 200 pg / ml.

[0068] Examples of suitable cell culture media include, but are not limited to, Eagle's Basal Medium, Minimum Essential Medium, Dulbecco’s Modified Eagle’s Medium (DMEM), Medium 199, Nutrient Mixtures Ham’s F-10 and Ham’s F- 12, McCoy’s 5A, Dulbecco’s MEM / F12, alpha modified Minimal Essential Medium (alphaMEM), Roswell Park Memorial Institute Media 1640 (RPM1 1640) and Iscove’s Modified Dulbecco’s Medium (IMDM). In one embodiment, the cell culture medium is DMEM.

[0069] Conventionally, adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs are maintained in cell culture using media supplemented with at least about 1% to 15% (v / v) serum, generally FBS, also known as fetal calf serum (FCS). The cell culture medium may be supplemented with FBS in a concentration from about 1% (v / v) to about 15% (v / v), about 3% (v / v) to about 14% (v / v), about 6% (v / v) to about 13% (v / v), about 8% (v / v) to about 12% (v / v), about 9% (v / v) to about 12% (v / v), more preferably, the FBS concentration may be 10% (v / v). In one embodiment the cell culture media is DMEM with 10% (v / v) FBS.

[0070] The term “chemically defined media” describes media comprising partly or fully defined components that are prepared artificially. They contain a balanced salt solution with a specific pH value and osmotic pressure designed for immediate survival of cells. Chemically defined media are typically entirely free of animal-derived components and can be supplemented with suitable additives supporting prolonged survival of the cell culture. In one embodiment, the chemically defined media do not contain and are not supplemented with a serum. In one embodiment, the chemically defined media do not contain and are not supplemented with FCS.

[0071] In one embodiment, the cell culture media used in the method of the present invention is xeno-free. Xeno-free media can contain human serum-derived components, but no components from animals other than humans. For avoidance of doubt, xeno-free culture medium is free of animal serum. Examples of xeno-free media include, but are not limited to, Cellartis DEF-CS 500 (Takara), X-VIVO 10, X-VIVO 15 and X-VIVO 20 (Lonza), mTeSR™1 , MesenCult™-ACF Plus and MesenCult™-ACF Plus (Stemcell Technologies), Human EpiVita Xeno-Free Growth Medium and HDF Growth Medium Xeno-free (Cell Applications), StemXVivo Serum-Free Human MSC Expansion Media (R&D systems), StemPro (Thermo Fisher Scientific), Stemline (MilliporeSigma) and RoosterBasal-MSC-CC (RoosterBio). Preferably, the xeno-free cell culture medium is RoosterBasal-MSC-CC (RoosterBio).

[0072] The cell culture medium used in the method of the present invention can also contain commercially available additives for xeno-free media. Examples of commercially available additives for xeno-free media include but are not limited to, Recombumin (Albumedix), CellPrime rlnsulin and CellPrime rAlbumin AF-S (MilliporeSigma), Fibronectin XF (Primorigen Biosciences), Optiferrin (InVitria), PLTMax (Mill Creek Life Sciences), B-27 Supplement, XenoFree (Thermo Fisher Scientific), KnockOut™ Serum Replacement, XenoFree (Thermo Fisher Scientific) and RoosterBooster-MSC-CC (RoosterBio).

[0073] Preferably, the additive to xeno-free culture medium is RoosterBooster-MSC-CC and in a further embodiment the preferred combination of xeno-free medium with additive is RoosterBasal-MSC-CC in combination with RoosterBooster-MSC-CC.

[0074] The cell culture medium used in the method of the present invention can also contain human derived additives such as human serum, human platelet cell lysate, recombinant human albumin, recombinant human transferrin or a combination of recombinant insulin and transferrin with selenium and ethanolamine albumin.

[0075] The cell culture medium used in the method of the present invention can also contain known serum replacements. The serum replacement can be, for example, fatty acid, collagen precursor, trace element, 2-mercaptoethanol or 3'-thiol glycerol, platelet-rich plasma, or those appropriately containing serum equivalents.

[0076] In the method of the present invention the adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs are cultured in a three-dimensional (3D) culture. A 3D culture refers to an artificially created environment in which cells are permitted to grow or interact with their surroundings in all three dimensions. Embodiments of 3D cultures include, but are not limited to, hydrogels, spheroids, clusteroids and microcarriers. Preferably, the 3D culture is performed on microcarriers. More preferably, the 3D culture is performed on microcarriers in a bioreactor.

[0077] As used herein, the term “bioreactor” refers to any vessel suitable for the growth of a cell culture. According to the disclosure the bioreactor is a vessel, closed at the top and bottom and connected with various pipes and valves. The vessel can be cylindrical, ranging in size from liters to cubic meters and can be made of glass or stainless steel. The internal conditions of the bioreactor, including but not limited to, pH and temperature, can be controlled during the culturing period. Examples of such bioreactors include, but are not limited to, a stirred-tank bioreactor, a plug flow bioreactor, a continuous stirred-tank bioreactor, and a stationary-bed bioreactor. A stirred-tank bioreactor is equipped with a stirring mechanism, such as a mechanical stirrer or an agitator, that keeps the contents of the bioreactor in motion and ensures that they are well-mixed. In a stirred-tank bioreactor the contents predominantly move in an axial direction. A stirred-tank bioreactor is typically operated in the batch or fed batch mode.

[0078] Various stirred-tank bioreactors are commercially available. Examples of commercially available stirred-tank bioreactors include, but are not limited to, Univessel® SU bioreactor manufactured by Sartorius, DASbox bioreactor manufactured by Eppendorf, HyPerforma™ DynaDrive manufactured by Thermo Fisher Scientific, BioBLU® Bioreactor manufactured by Eppendorf, Xcellerex bioreactors by Cytiva, Allegro™ STR Bioreactor by Pall Corporation and Mobius® Single-Use Bioreactors by Merck Millipore (MilliporeSigma). Preferably, the stirred-tank bioreactor is a Univessel® SU bioreactor manufactured by Sartorius. The Univessel® SU bioreactor is a disposable polycarbonate bioreactor, comprising several polycarbonate ports for media addition, sampling, harvesting as well as spare ports for insertion of classical sensors or other equipment. It further comprises 3-blade segment impellers, performing tasks such as mixing, aeration, heat and mass transfer within the vessel and an L-sparger located at the bottom of the bioreactor, which is used to introduce gas, typically oxygen, into a liquid medium in a bioreactor.

[0079] In the method of the present invention bioreactors with different volumes can be used. The bioreactor volume can range from 0.1 L - 100L, 0.1 L - SOL, 0.1 L - 60L, 0.1 L - 40L, 0.1 L - 20L, 0.1 L- 10L, 0.1 L - 8L, 0.1 L - 6L, 0.1 L - 4L, 0.1 L - 2L, 0.1 L - 1 L or 0.1 L - 0.5L. In one embodiment, cells are cultured in a bioreactor volume of 0.2L. In another embodiment, cells are cultured in a bioreactor volume of 2L.

[0080] Cell culture methods as provided in the present invention can be transitioned to larger production scales by using a “scale-up” process. The term “scale-up” refers to the process of increasing the production capacity of a bioreactor from an initially used smaller volume to a larger volume. By the process of scaling-up the bioreactor volume can be increased from a smaller volume of 0.1 L to 2L to a larger volume of 10L - 50L, 20L - 40L or 25L - 35L. In one embodiment the scale-up comprises cell migration via bead-to-bead transfer, wherein fresh microcarriers are provided into the cell culture vessel, preferably the bioreactor, and some cells spontaneously migrate from a cell- populated microcarrier to a fresh microcarrier that provides greater surface area for cell growth. In one embodiment, the ratio of cell-populated microcarrier to fresh microcarrier is 1 :2 to 1 :12, preferably the ratio of cell-populated microcarrier to fresh microcarrier is 1 :3 to 1 :10 or 1 :3 to 1 :8 or 1 :3 to 1 :6, more preferably the ratio of cell-populated microcarrier to fresh microcarrier is 1 :3 to 1 :5 and most preferably the ratio of cell-populated microcarrier to fresh microcarrier is 1 :4. In another embodiment the scale-up comprises harvesting cells followed by re-inoculation of single cells in larger scale bioreactors containing microcarriers.

[0081] The term microcarrier describes a small, typically spherical particle that provides a support matrix enabling attachment of adherent cells to form cell-microcarrier complexes suspended in cell culture medium.

[0082] Microcarriers can be made of a number of different materials including DEAE-dextran, glass, polystyrene, acrylamide, collagen, and alginate.

[0083] The microcarrier core refers to the central part or the structural foundation of a microcarrier particle and comprises the inner material of the microcarrier around which other layers or coatings may be applied. In one embodiment the microcarrier core is selected from the group consisting of cellulose, glass fiber, a synthetic polymer, a ceramic particle, a matrigel, an extracellular matrix component, collagen, poly L lactic acid, dextran, an inert metal fiber, silica, natron glass, borosilicate glass, chitosan, and a vegetable sponge. In a preferred embodiment the microcarrier comprises a synthetic polymer core. In one embodiment the synthetic polymer is selected from the group consisting of a polyester, a polypropylene, a polyalkylene, a polyfluorochloroethylene, a polyvinyl chloride, a polyvinyl fluoride resin, a polystyrene, a polysulfone, a polyurethane, or a polyethyene terephthalate. Preferably, the microcarrier comprises a polystyrene core.

[0084] In one embodiment, the microcarrier comprises a coating. A microcarrier coating refers to a layer of material applied to the core of a microcarrier to improve its compatibility with cells, promote cell attachment, and enhance cell growth. In a preferred embodiment the microcarrier comprises a glycoprotein coating. A glycoprotein refers to a class of proteins comprising carbohydrate groups attached to the polypeptide chain. In one embodiment, the microcarrier can be coated with one or more glycoproteins selected from the group consisting of fibronectin, vitronectin, chondronectin, and laminin. Preferably, the microcarrier is coated with vitronectin.

[0085] In one embodiment, the microcarrier is porous, i.e. it comprises a network of small pores or cavities throughout its structure which provides additional surface area for cell attachment and allows cells to migrate inside the microcarrier. In another embodiment, the microcarrier is macroporous. In still another embodiment, the microcarrier is non-porous with a solid surface. Preferably, the microcarrier is non-porous with a solid surface. More preferably, the microcarrier is made of a polystyrene core, coated with vitronectin and is non-porous with a solid surface. Various microcarrier are commercially available. Examples include, but are not limited to, Synthemax II microcarriers, low concentration (LC) Synthemax II microcarriers and Corning® Enhanced Attachment microcarriers (CEA) (all from Corning), Cytodex microcarriers (Cytiva), SoloHill Plastic, Plastic Plus microcarriers, ProNectin™ F and Hillex™ microcarriers (all from Sartorius) and GEM microcarriers (Avantor). Preferably, the microcarrier is a LC Corning™ Synthemax II microcarrier, which is a microcarrier with a polystyrene core which is coated with vitronectin and which is non-porous with a solid surface.

[0086] In one embodiment the adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, are cultured in a culture medium comprising microcarriers in a concentration range between 15 g / L and 25 g / L, between 17 g / L and 25 g / L, between 17 g / L and 24 g / L, between 17 g / L and 23 g / L or between 16 g / L and 22 g / L. In one embodiment the cells are cultured in a culture medium comprising microcarriers in a concentration of 16 g / L. In one embodiment the cells are cultured in a culture medium comprising microcarriers in a concentration of 22 g / L.

[0087] The term inoculating is used herein to refer to the process of introducing a defined number of adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, on a solid surface. In the context of the present invention the term solid surface refers to the surface of a microcarrier and is given as cm2.

[0088] In an embodiment, adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, are inoculated at a density of between 1 ,000 and 6,000 cells / cm2of the microcarrier, between 2,000 and 5,000 cells / cm2of the microcarrier, between 2,500 and 4,500 cells / cm2of the microcarrier or between 3,000 and 4,000 cells / cm2of the microcarrier. In a preferred embodiment, cells are inoculated at a density of 3,500 cells / cm2of the microcarrier.

[0089] During the culturing of adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, the cell culture is agitated, i.e. the cell culture medium and the microcarriers with the cells attached thereto are mechanically mixed or stirred. The term agitation speed refers to the speed at which the impeller (the rotating blade or paddle) rotates which is measured in revolutions per minute (rpm).

[0090] The term cell attachment is used herein to refer to the process of attaching adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs to the microcarriers. Cells attach to microcarriers through a series of interactions between the cell surface and the microcarrier's surface.

[0091] In one embodiment, the cells are detached from the microcarriers at the end of the culturing period. Optionally, the cells are first washed (e.g. 2-3 times), e.g. with a phosphate-buffered saline solution or comparable solution, before the cells are detached from the microcarriers.

[0092] The term cell detachment is used herein to refer to the process of detaching adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, from the microcarriers. Cells can be detached from microcarriers using different methods including, but not limited to, application of digestion enzymes, mechanical methods such as agitation or pipetting, and application of chelating agents or commercially available detachment solutions such as recombinant trypsin in combination with the chelating agent EDTA, e.g. as present in TrypLE™ Select, or Cell Dissociation buffer, enzyme free available from Gibco. Preferably the cells are detached by using a digestion enzyme. Examples for digestion enzymes suitable for detaching cells or formulations thereof include, but are not limited to, trypsin, papain, elastase, hyaluronidase, collagenase type 1 , collagenase type 2, collagenase type 3, collagenase type 4, or dispase. In one embodiment, the adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs are detached by contacting them with a recombinant trypsin in combination with the chelating agent EDTA, such as present in TrypLE™ Select.

[0093] In one embodiment, cultured cells contacted with a recombinant trypsin in combination with the chelating agent EDTA, e.g. as present in TrypLE™ Select, can be detached from the microcarriers by agitating the microcarriers for a period of time with an agitation speed sufficient to release the adherent cells, more preferably stem cells, preferably MSCs and most preferably ASCs, from the microcarriers. The agitation speed sufficient to release the adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, from the microcarriers can be about 165 rpm to 205 rpm, about 175 rpm to 195 rpm or about 180 rpm to 190 rpm. The agitation speed sufficient to release the adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, from the microcarriers can be increased during the detachment process. Thus, the detachment can start with an initial agitation speed of about 165 rpm to 205 rpm, directly followed by a period of increased agitation speed of about 300 rpm to 340 rpm. The period of time for which the initial agitation speed is applied can be about 2 minutes to 7 minutes. The period of time for which the increased agitation speed is applied can be about 5 seconds to 25 seconds.

[0094] Said sequence of applying an initial agitation speed for a defined period of time, directly followed by a shorter period of an increased agitation speed for a defined period of time is repeated about 8 to 12 times during detachment. In one embodiment, the adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, detached from the microcarriers are filtered before being washed and concentrated. The term filtration refers to a process in which solid particles in a liquid are removed by the use of a filter that permits the liquid to pass through, but retains solid particles, depending on the pore size of the filter.

[0095] The term filtration is used herein to refer to the process of separating microcarriers from said detached adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, and cell culture media by using small sterile sieves such as cell strainers or by pumping bioreactor’s fluid contents through a harvest port into a system, where a filter traps and captures the microcarriers. The system can be designed with a disposable bag containing a built-in filter with a specific pore size that is small enough to prevent the microcarriers from passing through, but large enough to allow cells and culture media to pass. Once the cells are filtered out of the medium, they are subjected to downstream processing while the microcarriers are retained within said disposable bag. Said filter can have a pore size ranging from about 60pm to 120pm, about 70pm to 110pm, about 80pm to 100pm or about 85pm to 95pm. In a preferred embodiment, said filter has a pore size of 90pm. In one embodiment, the filter is a nylon strainer having a pore size ranging from about 60pm to 120pm, about 70pm to 110pm, about 80pm to 100pm or about 85pm to 95pm. In a preferred embodiment, said filter is a nylon strainer having a pore size of 90pm. Examples of systems designed for separation of microcarriers from cells and cell culture media include, but are not limited to, Flexsafe® RM Bags with Microcarrier Filtration manufactured by Sartorius, Spinner Flasks and Microcarrier Separation Units manufactured by Corning, Magnetic Separation Systems manufactured by Miltenyi Biotec, Acoustic Wave Separation Technology manufactured by Pall Corporation and Harvestainer Microcarrier Separation Systems such as the 3L or 12L Harvestainer BioProcess Container manufactured by Thermo Scientific™. Preferably, the system for filtration is a Harvestainer BioProcess Container manufactured by Thermo Scientific™.

[0096] In one embodiment, the filtered adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, are further concentrated. In one embodiment a system for separating cells based on their sedimentation characteristics, size and density is used for concentrating the cells. Said system can utilize the principle of counterflow-based centrifugation, wherein the fluid flow direction is opposite to the centrifugal force direction, creating a controlled environment where cells are retained while the supernatant (fluid) is removed. Said system comprises a spinning chamber and as the chamber rotates, the centrifugal force pushes cells or particles toward the outer edges of the chamber. Simultaneously, a controlled flow of fluid is pumped into the chamber in the opposite direction of the centrifugal force, which allows the removal of waste fluids or media while retaining the cells or particles within the chamber. Said system allows for the concentration of cells by removing excess fluid while keeping the cells in the chamber and further performs cell washing by introducing a washing buffer into the chamber. After concentration and washing, the cells can be collected by reversing the flow direction or adjusting the centrifugation parameters. Examples for counterflow centrifugation systems for concentrating cells include, but are not limited to, Ksep50® system manufactured by Sartorius, Elutra™ Cell Separation System manufactured by Terumo BCT, MaxiMax™ Single-Use System manufactured by Repligen, Sefia Select™ system manufactured by Cytiva or Rotea Counterflow Centrifugation System manufactured by Thermo Fisher Scientific. Preferably, the system used for cell concentration is the Rotea Counterflow Centrifugation System manufactured by Thermo Fisher Scientific.

[0097] In one embodiment, the concentrated adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, are analyzed for critical quality attributes. Examples for critical quality attributes include, but are not limited to, viability, identity and potency of adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs.

[0098] The term viability refers to the proportion of living, healthy cells within a population of cells, determined by standard viability assays. The number of cells can be determined using automated cell counting systems. Examples for automated cell counting systems include, but are not limited to, Countess™ II FL Automated Cell Counter manufactured by Thermo Fisher Scientific, Cellometer® Auto 2000 manufactured by Nexcelom Bioscience, Luna™ II Automated Cell Counter manufactured by Logos Biosystems or NucleoCounter® NC-202™ manufactured by ChemoMetec. Preferably, the system for automated cell counting is NucleoCounter® NC-202™ manufactured by ChemoMetec. Various methods to assess viability of cells are known to the person skilled in the art. Examples for those methods include, but are not limited to, a dual-dye fluorescence-based assay using the fluorescent dyes acridin orange and propidium iodide (AO / PI), Trypan Blue Exclusion assay, MTT / MTS assay, assays using flow cytometry (e.g. Annexin V / PI staining, propidium iodide (PI) staining or Zombie Aqua™ staining), assays using microscopy (e.g. fluorescein diacetate (FDA) / PI double staining) or LDH release assay. Preferably, the method for assessing viability of cells is a dual-dye fluorescence-based assay using the fluorescent dyes acridin orange and propidium iodide (AO / PI) comprised in a NC-202 cassette, used with the NucleoCounter® NC-202™ manufactured by ChemoMetec. The viability can be expressed as a percentage of living cells relative to the total number of cells. In one embodiment the viability of the obtained adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, at the end of the culture is in the range of 70% to 100%, 75% to 95% or 80% to 90%. Preferably, the viability of obtained adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, at the end of the culture is at least 90%.

[0099] The term identity of cells refers to the specific characteristics and features that define a particular type of cell. In particular, identity of cells refers to specific characteristics and features that define adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, in particular the expression of the surface markers. A preferred assay for measuring the expression of surface markers is flow cytometry. Preferably, the population of MSCs may be characterized in that at least about 70%, at least about 80%, at least about 90% or at least about 95% of the population of cells express all of CD44, CD73, CD90 and CD105. Preferably, the population of ASCs may be characterized in that at least about 70%, at least about 80%, at least about 90% or at least about 95% of the population of cells express all of CD29, CD73, CD90 and CD105.

[0100] The term potency refers to the measurement of a biological activity of a cell population, in particular a biological activity which is relevant for the medical use of the adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs. It can refer to the ability of a substance to produce a desired biological effect or response in the cells within the cell culture. In one embodiment, potency refers to the ability of MSCs, in particular ASCs, to secrete the indoleamine 2,3-dioxygenase (IDO)-derived tryptophan metabolite kynurenine after induction with IFN- y. In one embodiment the ability of MSCs, in particular ASCs, to secrete kynurenine after induction with IFN-y is determined using high performance liquid chromatography (HPLC).

[0101] The yield of cells refers to the total number of adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, harvested from the culture at the end of the culture. The end of the culture herein refers to the point in time after the last step of concentrating the adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs.

[0102] In the following, the present invention will be described for a method using a medium comprising serum and for a method using a xeno-free medium comprising a serum replacement.

[0103] Serum-containing medium

[0104] In one embodiment the adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs are cultured in a medium comprising serum, preferably comprising FBS, more preferably comprising 10 % (v / v) FBS. In a preferred embodiment the adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs, are cultured in DMEM comprising serum, preferably comprising FBS, more preferably comprising 10 % (v / v) FBS.

[0105] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in a culture medium comprising serum for at least 10 to 20 days, 1 1 to 19 days, 12 to 18 days, 13 to 17 days or 14 to 16 days. In one embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in a culture medium comprising serum for 16 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in a culture medium comprising serum for 15 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in a culture medium comprising serum for 14 days.

[0106] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising FBS for at least 10 to 20 days, 11 to 19 days, 12 to 18 days, 13 to 17 days or 14 to 16 days. In one embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising FBS for 16 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising FBS for 15 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising FBS for 14 days.

[0107] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising 10 % (v / v) FBS for at least 10 to 20 days, 1 1 to 19 days, 12 to 18 days, 13 to 17 days or 14 to 16 days. In one embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising 10 % (v / v) FBS for 16 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising 10 % (v / v) FBS for 15 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising 10 % (v / v) FBS for 14 days. In one embodiment the ASCs, are cultured in a culture medium comprising serum for at least 10 to 20 days, 1 1 to 19 days, 12 to 18 days, 13 to 17 days or 14 to 16 days. In one embodiment, the ASCs, are cultured in a culture medium comprising serum for 16 days. In another embodiment, the ASCs are cultured in a culture medium comprising serum for 15 days. In another embodiment, the ASCs are cultured in a culture medium comprising serum for 14 days.

[0108] In one embodiment the ASCs are cultured in DMEM comprising FBS for at least 10 to 20 days, 1 1 to 19 days, 12 to 18 days, 13 to 17 days or 14 to 16 days. In one embodiment, the ASCs are cultured in DMEM comprising FBS for 16 days. In another embodiment, the ASCs are cultured in DMEM comprising FBS for 15 days. In another embodiment, the ASCs are cultured in DMEM comprising FBS for 14 days.

[0109] In one embodiment the ASCs are cultured in DMEM comprising 10 % (v / v) FBS for at least 10 to 20 days, 11 to 19 days, 12 to 18 days, 13 to 17 days or 14 to 16 days. In one embodiment, the ASCs are cultured in DMEM comprising 10 % (v / v) FBS for 16 days. In another embodiment, the ASCs are cultured in DMEM comprising 10 % (v / v) FBS for 15 days. In another embodiment, the ASCs are cultured in DMEM comprising 10 % (v / v) FBS for 14 days.

[0110] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in a culture medium comprising serum and between 50% and 90% or 60% and 80% of medium is replaced every 3 to 4 days. In a preferred embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in a culture medium comprising serum and 75% of medium is replaced every 3 days. In a preferred embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in a culture medium comprising serum and 75% of medium is replaced every 4 days.

[0111] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising FBS and between 50% and 90% or 60% and 80% of medium is replaced every 3 to 4 days. In a preferred embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising FBS and 75% of medium is replaced every 3 days. In a preferred embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising FBS and 75% of medium is replaced every 4 days.

[0112] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising 10 % (v / v) FBS and between 50% and 90% or 60% and 80% of medium is replaced every 3 to 4 days. In a preferred embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising 10 % (v / v) FBS and 75% of medium is replaced every 3 days. In a preferred embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising 10 % (v / v) FBS and 75% of medium is replaced every 4 days.

[0113] In one embodiment the ASCs are cultured in a culture medium comprising serum and between 50% and 90% or 60% and 80% of medium is replaced every 3 to 4 days. In a preferred embodiment, the ASCs are cultured in a culture medium comprising serum and 75% of medium is replaced every 3 days. In a preferred embodiment, the ASCs are cultured in a culture medium comprising serum and 75% of medium is replaced every 4 days.

[0114] In one embodiment the ASCs are cultured in DMEM comprising FBS and between 50% and 90% or 60% and 80% of medium is replaced every 3 to 4 days. In a preferred embodiment, the ASCs are cultured in DMEM comprising FBS and 75% of medium is replaced every 3 days. In a preferred embodiment, the ASCs are cultured in DMEM comprising FBS and 75% of medium is replaced every 4 days.

[0115] In one embodiment the ASCs are cultured in DMEM comprising 10 % (v / v) FBS and between 50% and 90% or 60% and 80% of medium is replaced every 3 to 4 days. In a preferred embodiment, the ASCs are cultured in DMEM comprising 10 % (v / v) FBS and 75% of medium is replaced every 3 days. In a preferred embodiment, the ASCs are cultured in DMEM comprising 10 % (v / v) FBS and 75% of medium is replaced every 4 days.

[0116] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in a culture medium comprising serum and between 10 g / L and 30 g / L, between 15 g / L and 28 g / L, between 17 g / L and 27 g / L, between 18 g / L and 25 g / L, between 19 g / L and 24 g / L or between 20 g / L and 23 g / L of microcarriers. Preferably, the adherent cells, preferably stem cells and more preferably MSCs are cultured in a culture medium comprising serum and 22 g / L of microcarriers.

[0117] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising FBS and between 10 g / L and 30 g / L, between 15 g / L and 28 g / L, between 17 g / L and 27 g / L, between 18 g / L and 25 g / L, between 19 g / L and 24 g / L or between 20 g / L and 23 g / L of microcarriers. Preferably, the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising FBS and 22 g / L of microcarriers. In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising 10 % (v / v) FBS and between 10 g / L and 30 g / L, between 15 g / L and 28 g / L, between 17 g / L and 27 g / L, between 18 g / L and 25 g / L, between 19 g / L and 24 g / L or between 20 g / L and 23 g / L of microcarriers. Preferably, the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising 10 % (v / v) FBS and 22 g / L of microcarriers.

[0118] In one embodiment the ASCs are cultured in a culture medium comprising serum and the culture medium comprises between 10 g / L and 30 g / L, between 15 g / L and 28 g / L, between 17 g / L and 27 g / L, between 18 g / L and 25 g / L, between 19 g / L and 24 g / L or between 20 g / L and 23 g / L of microcarriers. Preferably, the ASCs are cultured in a culture medium comprising serum and the culture medium comprises 22 g / L of microcarriers.

[0119] In one embodiment the ASCs are cultured in DMEM comprising FBS and between 10 g / L and 30 g / L, between 15 g / L and 28 g / L, between 17 g / L and 27 g / L, between 18 g / L and 25 g / L, between 19 g / L and 24 g / L or between 20 g / L and 23 g / L of microcarriers. Preferably, the ASCs are cultured in DMEM comprising FBS and 22 g / L of microcarriers.

[0120] In one embodiment the ASCs are cultured in DMEM comprising 10 % (v / v) FBS and between 10 g / L and 30 g / L, between 15 g / L and 28 g / L, between 17 g / L and 27 g / L, between 18 g / L and 25 g / L, between 19 g / L and 24 g / L or between 20 g / L and 23 g / L of microcarriers. Preferably, the ASCs are cultured in DMEM comprising 10 % (v / v) FBS and 22 g / L of microcarriers.

[0121] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in a culture medium comprising serum and between 10 g / L and 30 g / L, between 15 g / L and 28 g / L, between 17 g / L and 27 g / L, between 18 g / L and 25 g / L, between 19 g / L and 24 g / L or between 20 g / L and 23 g / L of microcarriers for 10 to 20 days, 11 to 19 days, 12 to 18 days, 13 to 17 days or 14 to 16 days. In one embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in a culture medium comprising serum and 22 g / L of microcarriers for 16 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in a culture medium comprising serum and 22 g / L of microcarriers for 15 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in a culture medium comprising serum and 22 g / L of microcarriers for 14 days.

[0122] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising FBS and between 10 g / L and 30 g / L, between 15 g / L and 28 g / L, between 17 g / L and 27 g / L, between 18 g / L and 25 g / L, between 19 g / L and 24 g / L or between 20 g / L and 23 g / L of microcarriers for 10 to 20 days, 1 1 to 19 days, 12 to 18 days, 13 to 17 days or 14 to 16 days. In one embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising FBS and 22 g / L of microcarriers for 16 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising FBS and 22 g / L of microcarriers for 15 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising FBS and 22 g / L of microcarriers for 14 days.

[0123] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising 10 % (v / v) FBS and between 10 g / L and 30 g / L, between 15 g / L and 28 g / L, between 17 g / L and 27 g / L, between 18 g / L and 25 g / L, between 19 g / L and 24 g / L or between 20 g / L and 23 g / L of microcarriers for 10 to 20 days, 11 to 19 days, 12 to 18 days, 13 to 17 days or 14 to 16 days. In one embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising 10 % (v / v) FBS and 22 g / L of microcarriers for 16 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising 10 % (v / v) FBS and 22 g / L of microcarriers for 15 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising 10 % (v / v) FBS and 22 g / L of microcarriers for 14 days.

[0124] In one embodiment the ASCs are cultured in a culture medium comprising serum and between 10 g / L and 30 g / L, between 15 g / L and 28 g / L, between 17 g / L and 27 g / L, between 18 g / L and 25 g / L, between 19 g / L and 24 g / L or between 20 g / L and 23 g / L of microcarriers for 10 to 20 days, 1 1 to 19 days, 12 to 18 days, 13 to 17 days or 14 to 16 days. In one embodiment, the ASCs are cultured in a culture medium comprising serum and 22 g / L of microcarriers for 16 days. In another embodiment, the ASCs are cultured in a culture medium comprising serum and 22 g / L of microcarriers for 15 days. In another embodiment, the ASCs are cultured in a culture medium comprising serum and 22 g / L of microcarriers for 14 days.

[0125] In one embodiment the ASCs are cultured in DMEM comprising FBS and between 10 g / L and 30 g / L, between 15 g / L and 28 g / L, between 17 g / L and 27 g / L, between 18 g / L and 25 g / L, between 19 g / L and 24 g / L or between 20 g / L and 23 g / L of microcarriers for 10 to 20 days, 11 to 19 days, 12 to 18 days, 13 to 17 days or 14 to 16 days. In one embodiment, the ASCs are cultured in DMEM comprising FBS and 22 g / L of microcarriers for 16 days. In another embodiment, the ASCs are cultured in DMEM comprising FBS and 22 g / L of microcarriers for 15 days. In another embodiment, the ASCs are cultured in DMEM comprising FBS and 22 g / L of microcarriers for 14 days.

[0126] In one embodiment the ASCs are cultured in DMEM comprising 10 % (v / v) FBS and between 10 g / L and 30 g / L, between 15 g / L and 28 g / L, between 17 g / L and 27 g / L, between 18 g / L and 25 g / L, between 19 g / L and 24 g / L or between 20 g / L and 23 g / L of microcarriers for 10 to 20 days, 1 1 to 19 days, 12 to 18 days, 13 to 17 days or 14 to 16 days. In one embodiment, the ASCs are cultured in DMEM comprising 10 % (v / v) FBS and 22 g / L of microcarriers for 16 days. In another embodiment, the ASCs are cultured in DMEM comprising 10 % (v / v) FBS and 22 g / L of microcarriers for 15 days. In another embodiment, the ASCs are cultured in DMEM comprising 10 % (v / v) FBS and 22 g / L of microcarriers for 14 days.

[0127] In one embodiment the adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs are cultured in a culture medium comprising serum and the cells are agitated intermittently during cell attachment. The term “intermittent agitation" or “agitated intermittently” describes an agitation profile, wherein periods of agitating and not agitating the cell culture are alternating. In one embodiment the agitation speed during cell attachment is about 70 rpm to 1 10 rpm, followed by a period of not agitating the cell culture.

[0128] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in a culture medium comprising serum, and the agitation speed is essentially kept constant during culturing. In one embodiment the essentially constant agitation speed during culturing is about 70 rpm to 1 10 rpm, 80 rpm to 100 rpm or 85 rpm to 95 rpm. Preferably, the constant agitation speed during culturing is 90 rpm. In one embodiment the adherent cells, preferably stem cells and more preferably MSCs, are cultured in a culture medium comprising serum and the cell culture is agitated constantly during culturing using an agitation speed of 90 rpm.

[0129] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising FBS, and the agitation speed is essentially kept constant during culturing. In one embodiment the essentially constant agitation speed during culturing is about 70 rpm to 1 10 rpm, 80 rpm to 100 rpm or 85 rpm to 95 rpm. Preferably, the constant agitation speed during culturing is 90 rpm. In one embodiment the adherent cells, preferably stem cells and more preferably MSCs, are cultured in DMEM comprising FBS and the cell culture is agitated constantly during culturing using an agitation speed of 90 rpm.

[0130] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising 10 % (v / v) FBS, and the agitation speed is essentially kept constant during culturing. In one embodiment the essentially constant agitation speed during culturing is about 70 rpm to 1 10 rpm, 80 rpm to 100 rpm or 85 rpm to 95 rpm. Preferably, the constant agitation speed during culturing is 90 rpm. In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in DMEM comprising 10 % (v / v) FBS and the cell culture is agitated constantly during culturing using an agitation speed of 90 rpm.

[0131] In one embodiment the ASCs are cultured in a culture medium comprising serum, and the agitation speed is essentially kept constant during culturing. In one embodiment the essentially constant agitation speed during culturing is about 70 rpm to 110 rpm, 80 rpm to 100 rpm or 85 rpm to 95 rpm. Preferably, the constant agitation speed during culturing is 90 rpm. In one embodiment the ASCs are cultured in a culture medium comprising serum and the cell culture is agitated constantly during culturing using an agitation speed of 90 rpm.

[0132] In one embodiment the ASCs are cultured in DMEM comprising FBS, and the agitation speed is essentially kept constant during culturing. In one embodiment the essentially constant agitation speed during culturing is about 70 rpm to 110 rpm, 80 rpm to 100 rpm or 85 rpm to 95 rpm. Preferably, the constant agitation speed during culturing is 90 rpm. In one embodiment the ASCs are cultured in DMEM comprising FBS and the cell culture is agitated constantly during culturing using an agitation speed of 90 rpm.

[0133] In one embodiment the ASCs are cultured in DMEM comprising 10 % (v / v) FBS, and the agitation speed is essentially kept constant during culturing. In one embodiment the essentially constant agitation speed during culturing is about 70 rpm to 110 rpm, 80 rpm to 100 rpm or 85 rpm to 95 rpm. Preferably, the constant agitation speed during culturing is 90 rpm. In one embodiment the ASCs are cultured in DMEM comprising 10 % (v / v) FBS and the cell culture is agitated constantly during culturing using an agitation speed of 90 rpm.

[0134] Xeno-free medium comprising a serum replacement

[0135] In one embodiment the adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs are cultured in a xeno-free culture medium comprising a serum replacement. In a preferred embodiment, the adherent cells, preferably stem cells, more preferably MSCs and most preferably ASCs are cultured in RoosterBasal-MSC- CC medium in combination with the additive RoosterBooster-MSC-CC. In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement for 1 to 12 days, 2 to 1 1 days, 3 to 10 days, 4 to 9 days or 5 to 8 days. In one embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement for 8 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement for 7 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement for 6 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno- free cell culture medium comprising a serum replacement for 5 days.

[0136] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC for 1 to 12 days, 2 to 1 1 days, 3 to 10 days, 4 to 9 days or 5 to 8 days. In one embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC for 8 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster- MSC-CC for 7 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC for 6 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC for 5 days.

[0137] In one embodiment the ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement for at least 1 to 12 days, 2 to 11 days, 3 to 10 days, 4 to 9 days or 5 to 8 days. In one embodiment, the ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement for 8 days. In another embodiment, the ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement for 7 days. In another embodiment, the ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement for 6 days. In another embodiment, the ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement for 5 days.

[0138] In one embodiment the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC for at least 1 to 12 days, 2 to 1 1 days, 3 to 10 days, 4 to 9 days or 5 to 8 days. In one embodiment, the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC for 8 days. In another embodiment, the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC for 7 days. In another embodiment, the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC for 6 days. In another embodiment, the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC for 5 days.

[0139] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement and the cell culture medium is not exchanged until the end of the cell culture. In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement and the cell culture is a batch cell culture. In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement and no anti-foam agent is added during the culture. In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement and no shear stress protectant is added to the culture. In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement and no anti-foam agent and no shear stress protectant is added to the culture. Shear stress protectants include, but are not limited to, Pluronic F68®, dextran, Ficoll 400, poloxamer 188, PEG11 , albumin, carboxymethyl cellulose, agar and gellan gum.

[0140] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC and the cell culture medium is not exchanged until the end of the cell culture. In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC and the cell culture is a batch cell culture. In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC and no anti-foam agent is added during the culture. In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC and no shear stress protectant is added to the culture. In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal- MSC-CC medium in combination with the additive RoosterBooster-MSC-CC and no anti-foam agent and no shear stress protectant is added to the culture.

[0141] In one embodiment the ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement and the cell culture medium is not exchanged until the end of the cell culture. In one embodiment the ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement and the cell culture is a batch cell culture. In one embodiment the ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement and no anti-foam agent is added during the culture. In one embodiment the ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement and no shear stress protectant is added to the culture. In one embodiment the ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement and no anti-foam agent and no shear stress protectant is added to the culture.

[0142] In one embodiment the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC and the cell culture medium is not exchanged until the end of the cell culture. In one embodiment the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC and the cell culture is a batch cell culture. In one embodiment the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC and no anti-foam agent is added during the culture. In one embodiment the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC and no shear stress protectant is added to the culture. In one embodiment the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC and no anti-foam agent and no shear stress protectant is added to the culture.

[0143] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement and between 10 g / L and 30 g / L, between 12 g / L and 28 g / L, between 14 g / L and 27 g / L, between 15 g / L and 25 g / L, between 16 g / L and 22 g / L, between 16 g / L and 20 g / L or between 16 g / L and 18 g / L of microcarriers. Preferably, the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement and 16 g / L of microcarriers.

[0144] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC comprising between 10 g / L and 30 g / L, between 12 g / L and 28 g / L, between 14 g / L and 27 g / L, between 15 g / L and 25 g / L, between 16 g / L and 22 g / L, between 16 g / L and 20 g / L or between 16 g / L and 18 g / L of microcarriers. Preferably, the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC comprising 16 g / L of microcarriers.

[0145] In one embodiment the ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement and between 10 g / L and 30 g / L, between 12 g / L and 28 g / L, between 14 g / L and 27 g / L, between 15 g / L and 25 g / L, between 16 g / L and 22 g / L, between 16 g / L and 20 g / L or between 16 g / L and 18 g / L of microcarriers. Preferably, the ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement and 16 g / L of microcarriers.

[0146] In one embodiment the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC comprising between 10 g / L and 30 g / L, between 12 g / L and 28 g / L, between 14 g / L and 27 g / L, between 15 g / L and 25 g / L, between 16 g / L and 22 g / L, between 16 g / L and 20 g / L, between 16 g / L and 18 g / L or between 16 g / L and 17 g / L of microcarriers. Preferably, the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC comprising 16 g / L of microcarriers.

[0147] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement and between 10 g / L and 30 g / L, between 12 g / L and 28 g / L, between 14 g / L and 27 g / L, between 15 and 25 g / L, between 16 g / L and 22 g / L, between 16 g / L and 20 g / L or between 16 g / L and 18 g / L of microcarriers for 1 to 12 days, 2 to 1 1 days, 3 to 10 days, 4 to 9 days or 5 to 8 days. In one embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement and 16 g / L of microcarriers for 8 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement and 16 g / L of microcarriers for 7 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement and 16 g / L of microcarriers for 6 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement and 16 g / L of microcarriers for 5 days.

[0148] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC comprising between 10 g / L and 30 g / L, between 12 g / L and 28 g / L, between 14 g / L and 27 g / L, between 15 g / L and 25 g / L, between 16 g / L and 22 g / L, between 16 g / L and 20 g / L or between 16 g / L and 18 g / L of microcarriers for 1 to 12 days, 2 to 1 1 days, 3 to 10 days, 4 to 9 days or 5 to 8 days. In one embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC comprising 16 g / L of microcarriers for 8 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC comprising 16 g / L of microcarriers for 7 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC comprising 16 g / L of microcarriers for 6 days. In another embodiment, the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal- MSC-CC medium in combination with the additive RoosterBooster-MSC-CC comprising 16 g / L of microcarriers for 5 days.

[0149] In one embodiment the ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement and between 10 g / L and 30 g / L, between 12 g / L and 28 g / L, between 14 g / L and 27 g / L, between 15 g / L and 25 g / L, between 16 g / L and 22 g / L, between 16 g / L and 20 g / L or between 16 g / L and 18 g / L of microcarriers for 1 to 12 days, 2 to 1 1 days, 3 to 10 days, 4 to 9 days or 5 to 8 days. In one embodiment, the ASCs are cultured in a xeno- free cell culture medium comprising a serum replacement and 16 g / L of microcarriers for 8 days. In another embodiment, the ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement and 16 g / L of microcarriers for 7 days. In another embodiment, the ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement and 16 g / L of microcarriers for 6 days. In another embodiment, the ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement and 16 g / L of microcarriers for 5 days.

[0150] In one embodiment the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC comprising between 10 g / L and 30 g / L, between 12 g / L and 28 g / L, between 14 g / L and 27 g / L, between 15 g / L and 25 g / L, between 16 g / L and 22 g / L, between 16 g / L and 20 g / L or between 16 g / L and 18 g / L of microcarriers for 1 to 12 days, 2 to 1 1 days, 3 to 10 days, 4 to 9 days or 5 to 8 days. In one embodiment, the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC- CC comprising 16 g / L of microcarriers for 8 days. In another embodiment, the ASCs are cultured in RoosterBasal- MSC-CC medium in combination with the additive RoosterBooster-MSC-CC comprising 16 g / L of microcarriers for 7 days. In another embodiment, the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC comprising 16 g / L of microcarriers for 6 days. In another embodiment, the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC comprising 16 g / L of microcarriers for 5 days.

[0151] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement, and the adherent cells, preferably stem cells and more preferably MSCs are continuously agitated during cell attachment. In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are continuously agitated during cell attachment at an agitation speed of about 70 rpm to 110 rpm, 80 rpm to 100 rpm or 85 rpm to 95 rpm. Preferably, the continuous agitation speed during cell attachment is 90 rpm. In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement and the adherent cells, preferably stem cells and more preferably MSCs are agitated continuously during cell attachment using a continuous agitation speed of 90 rpm.

[0152] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC, and the adherent cells, preferably stem cells and more preferably MSCs are continuously agitated during cell attachment. In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are continuously agitated during cell attachment at an agitation speed of about 70 rpm to 110 rpm, 80 rpm to 100 rpm or 85 rpm to 95 rpm. Preferably, the continuous agitation speed during cell attachment is 90 rpm. In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC and the adherent cells, preferably stem cells and more preferably MSCs are agitated continuously during cell attachment using a continuous agitation speed of 90 rpm.

[0153] In one embodiment the ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement, and the ASCs are continuously agitated during cell attachment. In one embodiment the ASCs are continuously agitated during cell attachment at an agitation speed of about 70 rpm to 110 rpm, 80 rpm to 100 rpm or 85 rpm to 95 rpm. Preferably, the continuous agitation speed during cell attachment is 90 rpm. In one embodiment the ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement and the ASCs are agitated continuously during cell attachment using a continuous agitation speed of 90 rpm. In one embodiment the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC, and the ASCs are continuously agitated during cell attachment. In one embodiment the ASCs are continuously agitated during cell attachment at an agitation speed of about 70 rpm to 110 rpm, 80 rpm to 100 rpm or 85 rpm to 95 rpm. Preferably, the continuous agitation speed during cell attachment is 90 rpm. In one embodiment the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC and the ASCs are agitated continuously during cell attachment using a continuous agitation speed of 90 rpm.

[0154] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement and the agitation speed is increased during culturing. In one embodiment said agitation speed starts from an initial agitation speed in the range of about 70 rpm to 110 rpm, 80 rpm to 100 rpm or 85 rpm to 95 rpm and is increased to an agitation speed in the range of about 140 rpm to 180 rpm, 150 rpm to 170 rpm or 155 rpm to 165 rpm during culturing. In one embodiment the agitation speed is increased daily by 10 rpm or 15 rpm. Preferably, the adherent cells, preferably stem cells and more preferably MSCs are cultured in a xeno-free cell culture medium comprising a serum replacement and the initial agitation speed is 90 rpm and is increased to 165 rpm during culturing.

[0155] In one embodiment the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC and the agitation speed is increased during culturing. In one embodiment said agitation speed starts from an initial agitation speed in the range of about 70 rpm to 110 rpm, 80 rpm to 100 rpm or 85 rpm to 95 rpm and is increased to an agitation speed in the range of about 140 rpm to 180 rpm, 150 rpm to 170 rpm or 155 rpm to 165 rpm during culturing. In one embodiment the agitation speed is increased daily by 10 rpm or 15 rpm. Preferably, the adherent cells, preferably stem cells and more preferably MSCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC and the initial agitation speed is 90 rpm and is increased to 165 rpm during culturing.

[0156] In one embodiment ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement and the agitation speed is increased during culturing. In one embodiment said agitation speed starts from an initial agitation speed in the range of about 70 rpm to 110 rpm, 80 rpm to 100 rpm or 85 rpm to 95 rpm and is increased to an agitation speed in the range of about 140 rpm to 180 rpm, 150 rpm to 170 rpm or 155 rpm to 165 rpm during culturing. In one embodiment the agitation speed is increased daily by 10 rpm or 15 rpm. Preferably, the ASCs are cultured in a xeno-free cell culture medium comprising a serum replacement and the initial agitation speed is 90 rpm and is increased to 165 rpm during culturing.

[0157] In one embodiment the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC and the agitation speed is increased during culturing. In one embodiment said agitation speed starts from an initial agitation speed in the range of about 70 rpm to 1 10 rpm, 80 rpm to 100 rpm or 85 rpm to 95 rpm and is increased to an agitation speed in the range of about 140 rpm to 180 rpm, 150 rpm to 170 rpm or 155 rpm to 165 rpm during culturing. In one embodiment the agitation speed is increased daily by 10 rpm or 15 rpm. Preferably, the ASCs are cultured in RoosterBasal-MSC-CC medium in combination with the additive RoosterBooster-MSC-CC and the initial agitation speed is 90 rpm and is increased to 165 rpm during culturing.

[0158] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. The invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing a claimed invention, from a study of the drawings, the disclosure, and the dependent claims. The detailed description is merely exemplary in nature and is not intended to limit application and uses. The following examples further illustrate the present invention without, however, limiting the scope of the invention thereto. Various changes and modifications can be made by those skilled in the art on the basis of the description of the invention, and such changes and modifications are also included in the present invention. EMBODIMENTS

[0159] Some embodiments of the present invention relate to:

[0160] 1 . A method of culturing adherent cells in a three-dimensional culture, the method comprising the steps of:

[0161] (a) obtaining cryopreserved adherent cells;

[0162] (b) thawing said cryopreserved adherent cells, thereby providing thawed adherent cells;

[0163] (c) directly inoculating said thawed adherent cells in a cell culture medium comprising microcarriers such that the adherent cells attach to the microcarriers; and

[0164] (d) culturing said adherent cells on said microcarriers.

[0165] 2. The method according to item 1 , wherein the microcarriers comprise a synthetic polymer core.

[0166] 3. The method according to item 2, wherein the synthetic polymer core is a polystyrene core.

[0167] 4. The method according to any of items 1 to 3, wherein the microcarriers comprise a surface coating.

[0168] 5. The method according to item 4, wherein the surface coating is a glycoprotein coating.

[0169] 6. The method according to item 5, wherein the glycoprotein coating is a vitronectin coating.

[0170] 7. The method according to any one of items 1 to 6, wherein the culture medium comprises 12 g / L to 25 g / L of said microcarriers.

[0171] 8. The method according to any one of items 1 to 7, wherein the adherent cells are cultured on said microcarriers in a bioreactor, preferably a stirred-tank bioreactor.

[0172] 9. The method according to any one of items 1 to 8, wherein the adherent cells are inoculated on said microcarriers at a density between 2,000 cells / cm2and 5,000 cells / cm2.

[0173] 10. The method according to any one of items 1 to 9, further comprising detaching the adherent cells from the microcarriers by application of a digestion enzyme.

[0174] 11 . The method according to item 10, further comprising agitating the microcarriers during detachment for a period of time at a frequency and agitation speed sufficient to release the adherent cells from the microcarriers.

[0175] 12. The method according to any of items 10 to 11 , further comprising separating said detached adherent cells from said microcarriers by filtration, thereby obtaining filtered adherent cells.

[0176] 13. The method according to item 12, further comprising concentrating the filtered adherent cells by counterflow-based centrifugation. The method according to any one of items 1 to 13, wherein the adherent cells are stem cells, preferably mesenchymal stem cells (MSCs) and more preferably adipose-derived stem cells (ASCs). The method according to item 14, wherein at least 90% of a population of said ASCs are positive for the surface markers CD29, CD90, CD73 and CD105. The method according to any one of items 1 to 15, wherein the cell culture medium comprises serum, preferably the cell culture medium comprises 10% fetal bovine serum (FBS). The method according to item 16, wherein the culture medium comprises 18 g / L to 25 g / L of said microcarriers. The method according to item 16 or 17, wherein the adherent cells are agitated intermittently during cell attachment. The method according to any one of items 16 to 18, wherein the agitation speed is essentially kept constant during culturing. The method according to any one of items 16 to 19, wherein the medium is exchanged at least once during cell culture. The method according to item 20, wherein the medium is exchanged every 3 to 4 days during cell culture. The method according to any one of items 16 to 21 , wherein the adherent cells are cultured for 14 to 16 days. The method according to any one of items 16 to 22, wherein the number of obtained adherent cells at the end of the culture is increased 2 to 4-fold compared to the initial number of thawed adherent cells. The method according to any one of items 16 to 23, wherein the concentration of obtained adherent cells at the end of the culture is between 1 -3 x 105cells / mL. The method according to any one of items 16 to 24, wherein the viability of obtained adherent cells at the end of the culture is at least 90%. The method according to any one of items 1 to 15, wherein the cell culture medium is a xeno-free medium, preferably comprising a serum replacement. The method according to item 26, wherein the culture medium comprises 12 g / L to 18 g / L of said microcarriers. The method according to item 26 or 27, wherein the adherent cells are agitated continuously during cell attachment. The method according to any one of items 26 to 28, wherein the agitation speed is increased during culturing. The method according to any one of items 26 to 29, wherein no media exchanges are performed during cell culture. The method according to any one of items 26 to 30, wherein the adherent cells are cultured for 5 to 8 days. The method according to any one of items 26 to 31 , wherein the number of obtained adherent cells at the end of cell culture is increased 6 to 7-fold, compared to the initial number of thawed adherent cells. The method according to any one of items 26 to 32, wherein the concentration of obtained adherent cells at the end of the culture is between 3-5 x 105cells / mL. The method according to any one of items 26 to 33, wherein the viability of obtained adherent cells at the end of the culture is at least 90%. The method according to any one of items 8 to 34, wherein the production capacity of the bioreactor is increased, preferably wherein the production capacity of the bioreactor is increased by cell migration via bead-to-bead transfer. A method of culturing adipose-derived stem cells (ASCs) in a three-dimensional culture, the method comprising the steps of:

[0177] (a) thawing said ASCs, thereby providing thawed adherent cells;

[0178] (b) directly inoculating said thawed adherent cells in a cell culture medium comprising microcarriers such that the ASCs attach to the microcarriers; and

[0179] (c) culturing said adherent cells on said microcarriers in a bioreactor, wherein said microcarriers comprise a synthetic polymer core and a glycoprotein surface coating. A method of culturing adipose-derived stem cells (ASCs) in a three-dimensional culture, the method comprising the steps of:

[0180] (a) thawing said ASCs, thereby providing thawed adherent cells;

[0181] (b) directly inoculating said thawed adherent cells in a cell culture medium comprising microcarriers such that the ASCs attach to the microcarriers; and

[0182] (c) culturing said adherent cells on said microcarriers in a stirred-tank bioreactor, wherein said microcarriers comprise a polystyrene core and a vitronectin surface coating. MATERIAL AND METHODS

[0183] 1 . Culturing of ASCs using different culture medium formulations

[0184] 1.1. Bioreactor set up

[0185] Prior to preparation of ASCs and the different cell culture medium formulations, the 2L Univessel® SU bioreactor was set up according to the instructions of the manufacturer.

[0186] 1 .2. Inoculation of ASCs and culture procedures using DMAX medium with 10% FBS

[0187] For the serum containing medium, DMEM (1 X) + GlutaMAX™ was supplemented with 10% FBS. For a 2 L culture with 22 g / L of LC Synthemax II microcarriers, 44 g of microcarriers were weighed in a sterile 500 mL flask. Microcarriers were resuspended in 160 mL of DMAX with 10% FBS and added to the bioreactor containing 500 mL of DMAX with 10% FBS. To promote the addition, the flask was lifted to add the content by gravity. The following target levels were reached, before the bioreactor was inoculated with the cells: dissolved oxygen (DO)=40%, pH=7.2 and T=37°C.

[0188] Cell thawing was performed using a dry thawing system (e.g. Thaw STAR system) and thawing time was 2-3 minutes from the time of vial insertion. Thawed ASCs of four vials (25 million cells / vial in approximately 1 mL) were transferred to a conical tube containing 36 mL of pre-warmed DMAX with 10% FBS. Cells were centrifuged at 1200 g for 6 minutes at Room Temperature (RT). The supernatant was aspirated, and the cells were resuspended with 48 mL of DMAX medium with 10% FBS to a final concentration of approximately 2 x 106cell / mL. For inoculation of cells into the bioreactor, the temperature of the bioreactor was retained at 37°C during the inoculation step whereas agitation, the pH control and the DO control were stopped. The volume of cells corresponding to 55.44 million cells was calculated and prepared in a final volume of 40 mL DMAX medium with 10% FBS. For inoculation of said volume, a syringe was connected to the bioreactor and the volume of 40 mL of cells was added by gravity to the bioreactor. The final bioreactor volume during the attachment phase (initial 24h following inoculation) was 800 mL. The agitation speed was set to 90 rpm for the first 24h and during culture time the agitation speed was increased from 90 rpm to 100 rpm to 110 rpm. 24h after cell inoculation, 1.2 L of DMAX medium with 10% FBS pre-warmed at 37SC were added to the bioreactor to a final medium volume of 2 L. 75% medium exchange was performed every 3-4 days after cell inoculation (i.e. on day 4, day 7, day 10 / 11 , day 13 / 14). For medium exchange, pH and DO controllers were switched off and the agitation was stopped. Microcarriers were allowed to settle down for at least 60-90 minutes. DMAX medium with 10% FBS corresponding to 75% of volume in the bioreactor at 37°C was prewarmed in a warm beads bath. The exact volume of spent medium was removed and replaced with the correct volume of pre-warmed DMAX medium with 10% FBS.

[0189] 1 .3. Inoculation of ASCs and culture procedures using RoosterBio Sup medium

[0190] RoosterBasal™-MSC was supplemented with 2% RoosterBooster™-MSC-XF to obtain RoosterBio Sup medium. For a 2 L culture with 16 g / L of LC Synthemax II microcarriers 32 g of microcarriers were weighed in a sterile 500 mL flask. Microcarriers were resuspended in 150 mL of RoosterBio Sup medium and added to the bioreactor containing 500 mL of RoosterBio Sup medium. To promote the addition, the flask was lifted to add the content by gravity. The following target levels were reached, before the bioreactor was inoculated with the cells: dissolved oxygen (DO)=40%, pH=7.2 and T°=37°C.

[0191] Cell thawing was performed using a dry thawing system (e.g. Thaw STAR system of STEMCELL Technologies) and thawing time was 2-3 minutes from the time of vial insertion. Thawed ASCs of three vials (25 million cells / vial in approximately 1 mL) were transferred to a conical tube containing 27 mL of pre-warmed RoosterBio Sup medium. Cells were centrifuged at 1200 g for 6 minutes at Room Temperature (RT). The supernatant was aspirated, and the cells were resuspended by pipetting 36 mL of RoosterBio Sup medium to a final concentration of approximately 2 x 106cell / mL. For inoculation of the cells into the bioreactor, the temperature of the bioreactor was retained at 37°C during the inoculation step, whereas agitation, the pH control and the DO control were stopped. The volume of cells corresponding to 40.32 million cells was calculated and prepared in a final volume of 40 mL RoosterBio Sup medium. For inoculation of said volume, a syringe was connected to the bioreactor and the volume of 40 mL of cells was added by gravity to the bioreactor. The final bioreactor volume during the attachment phase (initial 24h following inoculation) was 800 mL. Agitation speed was set to 90 rpm for the first 24h and during the culture the agitation speed was increased by 10-15 rpm each day to a final agitation speed of 165 rpm. 24h after cell inoculation, 1 .2 L of RoosterBio Sup medium pre-warmed at 37SC were added to the bioreactor to a final volume of 2L.

[0192] 1 .3 Culture procedures for the comparison of different microcarriers in DMAX medium with 10% FBS

[0193] The 0.2L stirred-tank DASbox bioreactor by Eppendorf was set up according to the instructions of the manufacturer. For the analysis of the colonization of ASCs on different types of microcarriers (SoloHill Plastic Plus microcarriers manufactured by Sartorius, Corning Enhanced Attachment (CEA) microcarriers or LC Synthemax II microcarriers also manufactured by Corning), DMAX medium with 10% FBS was prepared as described in section 1 .2. The different types of microcarriers were prepared to achieve a final concentration of 16 g / L for a 0.2L culture. Thawed ASCs were inoculated as described in section 1 .2 and resuspended with DMAX medium with 10% FBS to a final cell concentration of approximately 2.3 x 104cells / mL. The agitation speed was set to 60 rpm for the first 24h and during culture time the agitation speed was increased from 60 rpm to 80 rpm to 100 rpm. Exchange of medium was performed as described in section 1 .2.

[0194] 1 .4 Culture procedures for the comparison of different microcarriers in RoosterBio Sup medium

[0195] Cell vials comprising ASCs at Passage 2 were thawed using a ThawStar® CFT2 automated thawing system (STEMCELL Technologies). ASCs were inoculated immediately after thaw in BioBLU® 0.3c SU Vessels (Eppendorf™), equipped with a pitched-blade 45° impeller, and controlled using the DASbox® mini bioreactor system. Cells were cultured at 37°C, pH level controlled at 7.2 and dissolved oxygen of 8.4% 02 (corresponding to 40% of air saturation). The capacity of ASC to expand in the stirred system was evaluated using both Plastic and Low Concentration (LC) Synthemax® II Corning® microcarriers (Sigma-Aldrich) at a final concentration of 16 g / L. ASCs, without previous seed train, were inoculated at a cell density of 4 x 103cell / cm2in a total volume of 100 mL of RoosterNourish™ medium. During the first 24 h of the cell attachment phase to the microcarriers, either a continuous stirring at 60 rpm or an intermittent stirring regime (agitation cycles of 2 min of stirring at 60 rpm and 58 min with agitation off) was performed, after which the bioreactors were continuously agitated at 60 rpm in a working volume of 200 mL. As culture time progressed, to minimize microcarrier aggregation, the bioreactor stirring rate was increased up to 130 rpm. No medium change was performed. On the harvest day, the cells were detached from the microcarriers surface using TrypLE™ Select (1 X), a chemically-defined xeno-free reagent, at 37°C with the following cycles of stirring: 5 min at 150 rpm followed by a pulse of 15 s at 260 rpm, for a total incubation time of 30 min. After harvesting, the cell / microcarrier suspension was recovered from the bioreactor and filtered through a cell strainer (sterile nylon mesh) with pore size of 70 m (Falcon) into a 225 mL conical centrifuge tube (Falcon). The cell suspension was then centrifuged at 300 g for 10 min at RT. After cell concentration and viability analysis using the NucleoCounter® NC-202™ automated cell counter (ChemoMetec), cells were cryopreserved in a solution containing 90% FBS and 10% DMSO.

[0196] 1 .5 ASC expansion in 2 L stirred-tank bioreactors

[0197] ASC were inoculated immediately after thawing in 2L Univessel® SU (Sartorius) stirred-tank bioreactor equipped with two 3-blade segment impeller (30° angle). The scale-up from 0.2 L to 2 L was performed so that the power input per unit volume (P / V) was maintained constant. The stirring rates (N) implemented at the 2 L scale were therefore estimated based on Equation 1 : where P: power input; V: bioreactor working volume; N: stirring rate; p: liquid density; Np: impeller power number; Di: impeller diameter.

[0198] Similarly to ASC expanded in 0.2 L stirred-tank bioreactors, cells were cultured at 37°C, pH level controlled at 7.2, and 40% of air saturation. During the first 24 h of the cell attachment phase to the microcarriers, the stirred-tank bioreactor was continuously agitated at 90 rpm, after which 1 L of RoosterNourish™ medium was added to the bioreactor vessel to reach a working volume of 2 L. To limit microcarrier aggregation, throughout culture time, the stirring speed was increased up to 165 rpm.

[0199] 2. Sampling of ASCs during the cell culture

[0200] For sampling, it was guaranteed that the cell culture was homogenous and no microcarrier deposits were formed in the bioreactor so that the sampling step was representative of the cell numbers present in the bioreactor. If needed, agitation speed was increased by 10 rpm during the sampling process. Using a portable LFH (laminar flow hood), a 30 mL syringe was connected to the sampling line of the bioreactor and the sampling line was unclamped and the desired sample volume (= 15 mL) removed. The bioreactor sample was used to characterize the bioreactor cell culture for the parameters “microcarrier colonization", “cell viability” and “total viable cell numbers”. 3. Cell counting

[0201] 10 mL of the bioreactor sample was taken, microcarriers were left to settle down, the supernatant was removed and the microcarriers were washed using 5 mL of Phosphate Buffered Saline (PBS). After removal of PBS, 3 mL of TrypLE Select CTS was added to the microcarriers and incubated for at least 15 minutes at 37°C. After 15-45 minutes incubation at 37°C, the equivalent volume of the respective medium was added. 1 mL of the homogenous cell / microcarrier solution was used for cell counting using a NucleoCounter® NC-202™. The number of population doublings (PDs) were calculated according to Equation 2: where Cx corresponds to the total cell numbers at the time point t, and CO corresponds to the live cell numbers at inoculation.

[0202] 4. Assessment of cell viability

[0203] For qualitative analysis of cell viability, cells attached to the microcarriers were stained with 20 pg / mL of fluorescein diacetate (FDA, Sigma-Aldrich) and 10 pg / mL with the DNA-binding dye propidium iodide (PI, Sigma- Aldrich). Cell-seeded microcarriers were then analyzed at the inverted fluorescence microscope (DMI6000, Leica). For adhesion analysis, ASC were thawed and seeded at 30.000 cell / cm2 in T-flasks. 24 h later, they were harvested, and % of cells attached was assessed using NC-202 cell counter.

[0204] Cell viability was assessed by using the fluorescent dyes acridine orange and propidium iodide (AO / PI) comprised in NucleoCounter® NC-202™ cassettes and measuring the number of live and dead cells was performed using the automated cell counting system NucleoCounter® NC-202™. The viability was expressed as a percentage of living cells relative to the total number of cells.

[0205] 5. Microcarrier colonization

[0206] Cell colonization on microcarriers was assessed by fluorescein diacetate (FDA) and propidium iodide (PI) dual fluorescence analysis. A solution of 20 pg / mL FDA and 10 pg / mL PI in 500 pL of PBS was prepared and 50pL of the FDA / PI solution added to 300 pL of the bioreactor sample. Incubation was performed for 30 seconds at FIT in the dark. The cell / microcarrier solution was transferred to an ultra-low attachment 24-well plate and image acquisition was performed at 5X and 10X magnification. Microcarrier colonization was quantified using the Imaged software by merging the FDA (live cells) and brightfield (microcarriers) channels. The percentage of colonized microcarriers was calculated by the operator according to the following Equation 3:

[0207] Q% / col ioni .zed j mi .crocarri .ers = n -umber of - - microcarriers with at least one cell attached to the microcarrier total number of microcarriers

[0208] A minimum of 3 images at 4X magnification and 200 microcarriers were analyzed per condition.

[0209] 6. Harvest of ASCs

[0210] Cell harvest was performed when the target cell concentration of approximately 1 -5 x 105cells / mL was reached. 1 L of TrypLE Select CTS and 0.5L of DMAX 10% FBS medium were pre-warmed at 37°C in a warmed beads bath. For cell harvest, pH and DO controllers were switched off and the agitation was stopped. Microcarriers were allowed to settle down for at least 45-60 minutes. The maximum volume of medium (without removing cell / microcarriers) was removed from the bioreactor. 1 L of PBS was added to the bioreactor and the agitation rate of the bioreactor was set to 90 rpm for 1 minute. Afterwards, agitation was stopped and microcarriers were allowed to settle down for at least 30-40 minutes. The maximum volume of PBS (without removing cell / microcarriers) was removed from the bioreactor. Next, 1 L of TrypLE Select CTS pre-warmed to 37°C was added to the bioreactor.

[0211] To initiate the cell harvesting protocol, the following agitation cycles required for microcarrier / cells dissociation were performed: bioreactor agitation for 5 minutes at 185 rpm, followed by a pulse of 15 seconds at 320 rpm. Usually nine of these agitation cycles, corresponding to 45 minutes of incubation time with TrypLE, were performed. At the end of these cycles, a 5 mL sample was taken from the bioreactor to check under the microscope if cell dissociation was successful. If cells were still attached to the microcarriers, the number of agitation cycles was extended up to 12 cycles.

[0212] Once cells were dissociated from the microcarriers, 0.5L of DMAX 10% FBS pre-warmed to 37°C was added to the bioreactor. Using a portable LFH, the Harvestainer™ adaptor (connected to the Harvestainer™ inlet port) was connected to the Univessel® SU dip tube to collect the cells. The Harvestainer™ outlet port was then connected to the Harvestainer™ / Flexboy® adaptor, which in turn was also connected to the 3000 mL Flexboy® bag for cell collection. The cell / microcarrier suspension was recovered through the harvest port by activating the peristaltic pump (set to 400 mL / min). No additional PBS wash of the bioreactor / Harvestainer™ was performed. During the cell / microcarrier suspension collection (~1 minute) to the Harvestainer™, the bioreactor agitation rate was kept at 100 rpm. The cell suspension was further processed using a Rotea GTS counterflow system, allowing the 1 .5 L of cell suspension containing the solution of TrypLE Select and DMAX 10% FBS to be concentrated in a bag containing a final volume of 12 mL. Standard centrifugation (300 x g for 10 min at RT) was used as control to concentrate the manufactured ASG prior to their cryopreservation. Finally, cells were counted using a NucleoCounter® NC-202™ and further cryopreserved in a liquid / vapor-phase nitrogen container in a freezing solution [90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO)], from Gibco®, ThermoFisher Scientific and WAK-Chemie Medical GmbH, respectively) at a cell concentration of 25 million cell / mL.

[0213] 7. Bead-to-bead transfer step

[0214] Following cell expansion to approximately 1x105cell / mL (typically on day 4 of cell culture), corresponding to a stage of cell expansion when cells are in the proliferating phase but with limited cell / microcarrier aggregation levels, the content of the Univessel0bioreactor (32 g of populated microcarriers) was transferred into a 3000 mL bag. Afterwards, 32 g of the populated microcarriers were either added to 128 g of empty microcarriers (1 :4 ratio of cell populated: fresh microcarriers) or to 288 g of empty microcarriers (1 :9 ratio of cell populated : fresh microcarriers). The bead-to-bead transfer step has been successfully implemented at 2 L scale using intermittent stirring to facilitate cell migration between full and empty microcarriers. The intermittent stirring was performed in 2 L Univessel0SU every 30 min for 2 min at 100 rpm for 24 h.

[0215] 8. Immunophenotyping of ASG

[0216] ASCs, immediately cryopreserved after expansion in the stirred-tank bioreactors, were thawed and characterized by their immunophenotype; positive for CD73, CD90, CD29 and CD105 (antibodies from Miltenyi Biotec acquired with MACSQuant® Analyzer 10 / Miltenyi Biotec).

[0217] 9. Measurement of IDO activity

[0218] ASG seeded in a 24-well plate (2x104cells / well) were either left untreated or stimulated with IFN-y (3 ng / mL). IDO activity was measured by determining both tryptophan and kynurenine concentrations on conditioned supernatants at different time points. A total of 100 L of conditioned supernatants were mixed with 100 L of buffer phosphate 50 mM and 25 pL of 2 M trichloroacetic acid. After centrifugation for 10 min at 15,600 g, 100 pL of supernatant was analyzed by HPLC (Agilent, USA) with the Waters Empower™ Software (Waters, USA).

[0219] 10. Immunosuppression assay with ASCs

[0220] ASCs were plated in a 24-well plate. PBMCs (106cells / well) were activated with the Pan-T cell Activation kit (microbeads coated with anti-CD3 / CD2 / CD28; Miltenyi Biotec) following the manufacturer’s instructions or left unstimulated and cultured with or without ASG (ratio ASC:PBMC, 1 :25) in contact conditions in a total volume of 1 mL of RPMI + 10% FBS. After 96h of stimulation, PBMCs were harvested and cell proliferation was determined by cell counting using NucleoCounter® NC-202 (ChemoMetec).

[0221] 11 . Statistical analysis

[0222] Statistical analyses were performed using GraphPad Prism 10. Statistical significance was determined by one-way analysis of variance (ANOVA) with Tukey's multiple comparison testData are represented as mean ± standard deviation (SD). *p < 0.05, **p < 0.01 , ***p < 0.001 , **“p < 0.0001 were considered significant.

[0223] RESULTS

[0224] 1 . Colonization of ASCs on different microcarriers

[0225] During ASC expansion in a 0.2L stirred-tank DASbox bioreactor using DMAX medium with 10 % FBS, microcarrier colonization on different types of microcarriers (SoloHill Plastic Plus microcarriers manufactured by Sartorius, Corning Enhanced Attachment (CEA) microcarriers or LC Synthemax II microcarriers also manufactured by Corning) was assessed as shown in Figure 1 A. The percentage of cells attached to the microcarriers during all days of cell culture was highest using LC Synthemax II microcarriers, independent of whether the applied agitation protocol was continuous or intermittent. Using LC Synthemax II microcarriers, the microcarrier colonization at the end of the cell culture (day 14) was about 90%, whereas CEA microcarriers only reached a microcarrier colonization of about 70%. Using Plastic Plus microcarriers, the maximum microcarrier colonization of about 60% was reached at day 5 of cell culture and could not be maintained. It was confirmed that the LC Synthemax II microcarriers have higher microcarrier colonization levels (70±4% vs 30±2% at 24 h after inoculation - Fig. 1 B) compared to Plastic microcarriers also when RoosterBio Sup medium was used.

[0226] These data suggest that LC Synthemax II microcarriers can support cell growth while rendering a significantly higher and more homogeneous microcarrier colonization in comparison to Plastic Plus and CEA microcarriers.

[0227] 2. Cell attachment and microcarrier colonization using RoosterBio Sup medium

[0228] During the process of ASC expansion in a 2L stirred-tank bioreactor supported by LC Synthemax II microcarriers using RoosterBio Sup medium, homogeneous attachment of cells was observed. Using microcarrier concentrations of 16 g / L and 22 g / L, microcarrier colonization along culture time is shown in Figure 2. The percentage of microcarrier surface colonized byASCs was increasing during culture, reaching a microcarrier colonization of about 80% at day 5 of cell culture, using microcarrier concentrations of both 16 g / L and 22 g / L. As no significant differences were observed throughout culture time regarding microcarrier colonization, it is concluded that both a microcarrier concentration of 16 g / L and 22 g / L equally support a homogeneous microcarrier colonization.

[0229] 3. Cell concentration and viability throughout ASC expansion using RoosterBio Sup medium

[0230] Cell concentration and viability throughout ASC expansion in a 2L stirred-tank bioreactor supported by LC Synthemax II microcarriers (16 g / L or 22 g / L) using RoosterBio Sup medium is shown in Figure 3 A. On day 5 of cell culture, cell concentrations with microcarrier concentrations of 16 g / L and 22 g / L were about 4 x 105cells / mL and viability of cells was close to 100 %. The cumulative population doublings (PDs) along cell culture duration are depicted in Figure 3 B and show equal population doublings of about 4 PDs on day 5 of cell culture. As no significant differences were observed throughout culture time regarding cell concentration, viability and number of PDs, it is concluded that both a microcarrier concentration of 16 g / L and 22 g / L equally support expansion of ASCs under the conditions described herein.

[0231] 4. Cell concentration and viability throughout ASC expansion using DMAX 10 % FBS medium

[0232] Cell concentration and viability throughout ASC expansion in a 2L stirred-tank bioreactor supported by LC Synthemax II microcarriers (22 g / L) in DMAX 10 % FBS medium is shown for two donors (DON A and B) in Figure 4. On day 14 of cell culture, an increase in cell concentration and cell viability was observed for two different donors throughout culture time. Therefore, it is concluded that a microcarrier concentration of 22 g / L using DMAX 10 % FBS medium supports expansion of ASCs under the conditions described herein.

[0233] 5. Bead-to-bead transfer

[0234] To evaluate the efficacy of the bead-to-bead transfer step in comparison with single cell re-inoculation strategy as scale-up approaches, ASC were cultured for 4 days in stirred-tank bioreactors, after which either 10% - B:to:B (1 :9) condition - or 20% - B:to:B (1 :4) condition - of the cell-seeded microcarriers were transferred to a new bioreactor vessel with 90% or 80% of fresh SL microcarriers, respectively, to a final microcarrier concentration of 16 g / L. While the bead-to-bead transfer strategy was applied when cell-seeded microcarriers reached approximately 1x105cell / mL (Day 4 of culture), so that cells would achieve their exponential growth phase while still retaining low microcarrier aggregation levels, the single cell re-inoculation approach was performed when the microcarriers reached their highest confluency levels, corresponding to a cell concentration of approximately 4x105cell / mL (Day 6 of culture). Of note, while a bead-to-bead transfer step implemented at a 1 :9 ratio of fulkempty beads resulted in 68% colonized microcarriers on the harvest day (Day 9), higher colonization levels were observed for both single cell re-inoculation strategy (92% on Day 12) and bead-to-bead transfer applied at a 1 :4 ratio (87% on Day 8) (Fig. 5A). While the bead-to-bead transfer step resulted in a cell harvest concentration of 3.2 x 105cell / mL or 3.9 x 105cell / mL after 4 days of expansion, whether a ratio of fulkempty bead of 1 :9 or 1 :4, respectively, was used, the single cell re-inoculation strategy resulted in a cell concentration at harvest after six days of 3.8 x 105cell / mL (Fig. 5B), supporting the feasibility of both scale-up strategies.

[0235] During the 8 to 12 days of overall cell culture in the bioreactors (considering both the parental and production bioreactors), cumulative PDs between 6.6 and 7.4 (bead-to-bead transfer at 1 :4 and 1 :9, respectively) and 8.1 (single cell re-inoculation strategy) were obtained (Fig. 6A). Importantly, although the higher colonization of microcarriers transferred at a higher fulkempty bead ratio (1 :4) led to approximately 20% higher cell concentrations at harvest compared to using a lower fulkempty beads ratio (1 :9), the latter strategy offers the advantage of enabling inoculation of larger production bioreactor volumes. Indeed, the bead-to-bead transfer step at ratios of 1 :4 and 1 :9 would enable inoculation of 10 L and 20 L bioreactors, respectively (using a 2 L parental bioreactor and retaining the microcarrier concentration of 16 g / L in the production bioreactor). Besides, while the single cell re-inoculation strategy would allow a production bioreactor (at 16 g / L and 3500 cell / cm2) to be inoculated with a working volume of 26.5 L considering an harvest efficiency of 74% (after microcarrier / cell separation and centrifugation - Fig. 6B), the bead-to-bead transfer step does not require cell / microcarrier dissociation prior to inoculating larger scale bioreactors and therefore minimizes the number of operations required to scale-up ASC production and has a shorter process time of 8-9 days vs 12 days.

[0236] Importantly, all conditions retained high cell viability after harvest (above 95% - Fig. 6C) and expression of the MSC characteristic surface markers CD29, CD90, CD73 and CD105 (above 99% - Fig. 6D), with no statistical differences observed relatively to cells cultured under static conditions.

[0237] 6. Counterflow centrifugation

[0238] A scalable protocol for cell harvesting from microcarriers in stirred culture systems was implemented, using a filtration bag (Harvestainer™) for cell separation from microcarriers and a counterflow centrifugation step to concentrate cells. Final cell recoveries relative to the sampling point are shown in Fig. 7. The counterflow centrifugation, applied to perform volume reduction and concentrate ASCs after harvest, resulted in similar recovery yields (67±4%) when compared with the standard and less scalable centrifugation protocol (75±4%). High cell viability (>96%) was obtained for all conditions under evaluation immediately before cell cryopreservation. All conditions resulted in expression of the surface markers CD29 / CD90 and CD73 / CD105 above 95% (Fig. 8B) and a cell diameter that ranged between 16 and 19 pm (Fig. 8C). The ability of ASCs to secrete the IDO-derived tryptophan metabolite kynurenine after induction with IFN-y, involved in suppression of T cell proliferation, was determined by HPLC. The concentration of kynurenine, quantified at 24 h, was similar for all conditions under evaluation (above 9 pM) (Fig. 8D). The same applies to the inhibition of lymphoproliferation (Fig. 8E). Hence, it could be shown that counterflow centrifugation which leverages a dynamic fluid barrier to concentrate cells in a gentle manner (Li et al. (2021) Cytotherapy 23: 774-786) could be used to wash and reduce the volume of ASCs recovered from the bioreactor without impacting cell viability and cell quality while ensuring a concentration factor of 125-fold (from 1500 mL to 12 mL).

Claims

CLAIMS1 . A method of culturing adherent cells in a three-dimensional culture, the method comprising the steps of:(a) thawing said adherent cells, thereby providing thawed adherent cells;(b) directly inoculating said thawed adherent cells in a cell culture medium comprising microcarriers such that the adherent cells attach to the microcarriers; and(c) culturing said adherent cells on said microcarriers.

2. The method according to claim 1 , wherein the microcarriers comprise a synthetic polymer core, preferably a polystyrene core.

3. The method according to any of claims 1 or 2, wherein the microcarriers comprise a surface coating, preferably a glycoprotein coating, more preferably a vitronectin coating.

4. The method according to any one of claims 1 to 3, wherein the culture medium comprises 12 g / L to 25 g / L of said microcarriers.

5. The method according to any one of claims 1 to 4, wherein the adherent cells are cultured on said microcarriers in a bioreactor, preferably a stirred-tank bioreactor.

6. The method according to any one of claims 1 to 5, wherein the adherent cells are inoculated on said microcarriers at a density between 2,000 and 5,000 cells / cm2.

7. The method according to any one of claims 1 to 6, wherein the adherent cells are stem cells, preferably mesenchymal stem cells (MSCs), more preferably adipose-derived stem cells (ASCs).

8. The method according to claim 7, wherein the adherent cells are ASCS and at least 90% of a population of said ASCs are positive for the surface markers CD29, CD90, CD73 and CD105.

9. The method according to any one of claims 1 to 8, wherein the cell culture medium comprises serum, preferably comprises 10% fetal bovine serum (FBS).

10. The method according to claim 9, wherein the culture medium comprises 18 g / L to 25 g / L of said microcarriers.11 . The method according to any one of claims 9 or 10, wherein the adherent cells are cultured for 14 to 16 days.

12. The method according to any one of claims 1 to 8, wherein the cell culture medium is a xeno-free medium.

13. The method of claim 12, wherein the cell culture medium comprises a serum replacement.

14. The method according to any of claims 12 or 13, wherein the culture medium comprises 12 g / L to 18 g / L of said microcarriers.

15. The method according to any one of claims 12 to 14, wherein the adherent cells are cultured for 5 to 8 days.

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