Pooled mesenchymal stromal cell-derived cells, cell-free preparations thereof

Abstract

The present invention is based on a mesenchymal stromal cell (MSC) pooling and cell passaging strategy that provides highly active master bank and working bank cell preparations that can be used directly or cultured and expanded after freezing and thawing to obtain either cell products or cell-free products, such as cell-free culture supernatants, that are therapeutically active and can be used in a variety of clinical applications. The present invention provides methods for pooling MSCs and MSC preparations or cell-free compositions obtained from such MSC preparations.

Description

[Technical Field]

[0001] The present invention is based on a mesenchymal stromal cell (MSC) pooling and cell passaging strategy that provides highly active master bank and working bank cell preparations that can be used directly or cultured and expanded after freezing and thawing to obtain either cell products or cell-free products, such as cell-free culture supernatants, that are therapeutically active and can be used in a variety of clinical applications. The present invention provides methods for pooling MSCs and MSC preparations or cell-free compositions obtained from such MSC preparations. [Background technology]

[0002] Human mesenchymal stromal cells (hMSCs) have been widely investigated in cell therapy. Specifically, MSCs likely exert their therapeutic activity by secreting factors related to chemoattraction, cell proliferation and differentiation, immunomodulation, angiogenesis, anti-apoptosis, anti-fibrosis, and even antimicrobial effects (1, 5-7). This has led to several promising preclinical studies and Phase I and II clinical trials. However, progress to Phase III and IV clinical trials, or even to marketing approval, has been significantly delayed. hMSC therapies tested to date have proven neither cost-effective nor competitive with best-practice therapies, often associated with inconsistent research results, as well as safety and efficacy issues (12). Technical obstacles (e.g., scale-up and cryopreservation) and issues surrounding hMSC biology, such as donor variability, functional senescence, and a wide variety of proposed mechanisms of action (MoA), further complicate the issue (13). Therefore, to obtain robust and valid clinical data, it is paramount to manufacture significant hMSC doses from reproducible clinical hMSC products that can be tested in large-scale randomized clinical trials. Furthermore, these products and their clinical evaluation require approval by the relevant regulatory authorities, which expects a thorough scientific approach addressing GMP-compliant manufacturing, comprehensive quality control, and rigorous preclinical efficacy and safety testing (13).

[0003] The issue of hMSC scale-up has been much debated, following the failure of large-scale Phase III clinical trials to meet clinical endpoints. One allogeneic hMSC therapeutic, Prochymal™, lacked efficacy when expanded in vitro to produce multiple clinical doses. In contrast, other allogeneic hMSC products, when expanded to smaller clinical doses, have reproducibly demonstrated efficacy in studies of steroid-refractory graft-versus-host disease (GvHD) (14). This suggests that hMSC production must be carefully balanced to produce a sufficient number of clinical doses and allow for few population doublings during ex vivo production.

[0004] Inconsistent clinical trial results may be due to donor-to-donor variability when hMSCs are produced from a single donor (15). To address this issue, the concept of hMSC pooling has been developed. As an example, a product called "MSC-FFM" was produced from pooled bone marrow (BM) mononuclear cells (MNCs) containing not only hMSC precursors but also alloreactive immune cells from eight healthy third-party donors (16). In addition to reducing donor variability, the alloimmune response was intended to generate immunologically primed hMSCs with a stronger immunosuppressive potency. Indeed, these cells have been reported to have beneficial effects in children and adults with severe steroid-refractory GvHD (17, 18). However, for the purpose of chronic wound healing, highly immunosuppressive hMSCs may not be the first choice. Another example of a pooled hMSC product is "Stempeucel™," which has been successfully evaluated in critical limb ischemia. Here, we established a donor master cell bank (MCB) of single-donor hMSCs after passage 1 (19). Next, we generated a working cell bank (WCB) by pooling hMSCs from three donors. This was then expanded for five additional passages before cryopreservation of the final product. While pooled "MSC-FFM" products require the establishment of a new pooled hMSC MCB from scratch, the concept of a single-donor "Stempeucel™" hMSC MCB allows for high batch-to-batch consistency, as recently demonstrated (20). However, replicative senescence during the five passages required to reach the final product formulation may impact the quality of the clinical product (21).

[0005] In addition to donor variability and large-scale expansion, issues with cryoinjury and administration have been argued to compromise the success of hMSC clinical applications. To facilitate manufacturing and delivery, hMSCs are typically cryopreserved as clinical products after ex vivo expansion, then transported to the patient's bedside and administered immediately after thawing. However, cryopreservation may affect clinical efficacy due to its association with heat shock response, reduced immunomodulatory and homing capabilities (27-29), and increased tissue factor expression (30).

[0006] Human mesenchymal stromal cells (hMSCs) are a promising source of cell-based therapies. However, progress to Phase III and IV clinical trials has been remarkably slow. We aimed to first develop a manufacturing concept that balances large-scale production of pooled hMSCs with minimal expansion time to mitigate donor variability and generate robust, valid clinical data, and second, to test key manufacturing and efficacy endpoints in clinically relevant indications such as wound healing. Summary of the Invention

[0007] In the context of the present invention, although the invention is not limited generally, an exemplary novel clinical-scale manufacturing concept has been developed, which involves pooling cells into a working cell bank from which an extrapolated number of 70,000 cells can be produced in just three passages to produce cells for a wound size of 1 cm. 2 1x10 6 This study contains six single-donor hMSC master cell banks capable of producing clinical doses of hMSCs. Pooled hMSC batches demonstrated high stability in key manufacturing indicators, including morphology, immunophenotype, proliferation, scratch wound healing, chemotactic migration, and angiogenesis support. Repeated topical hMSC administration significantly accelerated wound healing in a diabetic rat model by delivering defined growth factor cargoes (specifically, BDNF, EGF, G-CSF, HGF, IL-1α, IL-6, LIF, osteopontin, VEGF-A, FGF-2, TGF-β, PGE-2, and, after priming, IDO) at specific stages of wound repair, namely, inflammation, proliferation, and remodeling. Specifically, hMSCs mediated epidermal and dermal maturation and collagen formation, improved angiogenesis, and promoted cell infiltration. Kinetic analysis revealed the transient presence of hMSCs by day 4 (d4) and the dynamic recruitment of macrophages that infiltrated from the wound edge (d3) and base (d9) and eventually progressed to the wound apex by d11. In the wound, hMSCs mediated M2-like macrophage polarization that began on d4, peaked on d9, and then declined by d11.

[0008] This exemplary study of the present invention establishes a standardized, scalable pooled hMSC therapeutic that delivers a defined cargo of trophic factors, which is effective in diabetic wound healing by improving angiogenesis and the dynamic recruitment of M2-like macrophages. This decision-making study will now enable testing of pooled hMSCs as a treatment for impaired wound healing in large-scale randomized clinical trials.

[0009] Generally and briefly, therefore, the main aspects of the present invention can be described as follows.

[0010] In a first aspect, the present invention provides a method for generating a donor cell pool of expandable cells derived from bone marrow samples of multiple genetically non-identical bone marrow donors, comprising: (a) providing two or more bone marrow donor samples from genetically non-identical (or different) bone marrow donors; (b) separately passaging bone marrow-derived cells from each bone marrow donor sample of (a) into (separate) cell culture receptacles such as culture flasks or dishes, bioreactors, or 3D expansion and growth systems (p0); (c) culturing bone marrow-derived cells from each donor separately; (d) (p1) separately subculturing a portion of each culture of bone marrow-derived cells from (c) into a new cell culture receptacle such as a culture flask or dish; (e) culturing bone marrow-derived cells from each donor separately; (f) Passaging and pooling a portion of each culture of bone marrow-derived cells from (e) into a single cell culture receptacle to obtain a donor cell pool (p2); The present invention relates to a method, including:

[0011] In a second aspect, the present invention relates to working cell banks and / or clinical products produced by the methods of the present invention.

[0012] In a third aspect, the present invention relates to a preparation of clinical-grade pooled mesenchymal stromal cells (MSCs) produced by the method of the present invention.

[0013] In a fourth aspect, the present invention relates to a preparation of clinical-grade pooled mesenchymal stromal cells (MSCs) for use in treating a disease or condition in a subject.

[0014] In a fifth aspect, the present invention relates to a method for producing a therapeutically active composition, for example a composition containing cell-free growth factors, comprising carrying out a method according to the invention or providing a working cell bank and / or clinical product or preparation of any aspect of the invention, followed by culturing and / or expanding MSC material in cell culture and harvesting the composition into which the MSC material has secreted one or more cellular factors, to obtain the therapeutically active composition.

[0015] In a sixth aspect, the present invention relates to a therapeutically active composition containing one or more growth factors made by the method of the present invention.

[0016] In a seventh aspect, the present invention provides a pharmaceutical composition comprising a cell or cell-free material of any aspect of the invention or obtained or obtainable by any aspect of the invention. [Brief explanation of the drawings]

[0017] [Figure 1](A) Pooling concept: hMSCs were isolated from the bone marrow of six individual donors and expanded (passage p0). To initiate passage 1 expansion, single-donor hMSCs were then thawed and pooled at the beginning of passage 1 (pool 1) or expanded individually as single-donor hMSCs. These were then pooled at the beginning of passage 2 (pool 2) or passage 3 (pool 3), respectively. Pooled hMSCs at the end of passage 3 were cryopreserved and formulated as clinical products. (B) Master and Working Cell Bank concepts: At the end of passage 0, hMSCs from a single donor were cryopreserved as a single-donor MCB. Single-donor MCBs expanded at passage 1 were cryopreserved at harvest after passage 1 to form a WCB. Pool 2 hMSCs pooled at the beginning of passage 2 were cryopreserved at the end of passage 2 as a working cell bank (WCB pool 2). An aliquot from this WCB was thawed and expanded for one further passage, yielding a potential clinical product at the end of passage 3. These cells were thawed and used for all experiments. Generated by BioRender.com. [Figure 2] Schematic of the in vivo wound healing assay. (A) Diabetic ZDF rats were wounded and topically treated with hMSCs (1 x 10 cells per cm of wound) in dilute fibrin glue. Untreated wounds and acellular fibrin glue-treated wounds served as controls (generated by BioRender.com). (B) Table depicting animal allocation. Abbreviations: fresh = rescue-cultured MSCs for up to 2 days before administration to the wound; cryo = thawed MSCs immediately prior to application; 1x = single application on day 0; 3x = repeated applications on days d0, 4, and 8. [Figure 3]Figure 1 shows that all hMSC pools were characterized by similar proliferative potential, but the highest number of extrapolated clinical doses could be achieved with hMSCs from Pool 2. All pools exhibited similar functional properties. (A) Calculation of maximum achievable cell dose / manufacturing batch and (B) manufacturing cell dose / batch (1 x 10 hMSCs per cm wound size) for pooled hMSCs at either passage 1 (Pool 1), passage 2 (Pool 2), or passage 3 (Pool 3). (C) Flow cytometry characterization of binary (absent or present) hMSC markers. (D-H) Functional characterization of hMSC pools. Data are shown normalized to Pool 2. (D) hMSC-mediated inhibition of PHA-driven T cell proliferation. (E-H) Live cell imaging analysis of functional hMSC attributes: (E) proliferation; phase-regression confluence 96 h after seeding; (F) scratch wound healing; relative wound density 24 h after wounding; (G) tube network formation; hMSCs were seeded as a monolayer on top of fluorescently labeled endothelial cells (HUVECs). Tube length was assessed 48 h after seeding and calculated as a percentage of human adipose stromal cell-mediated tube formation; (H) chemotactic migration into medium supplemented with hPL in the bottom chamber. The number of migrated cells in the bottom well was normalized to the initial value in the top well. Serum-free medium served as a negative control. (E-H) Lowercase letters indicate experimental replicates performed by different operators on different days. All data are presented as individual experimental replicates and represent the mean ± SD. [Figure 4]Figures 1-3 show pilot studies to assess cryoinjury, dose titration, and systemic wound healing effects. (A) Representative images of wounded skin after a single topical treatment with either hMSCs in fibrin glue, acellular fibrin glue, or untreated on day 0 after wounding. (B) Reduction in wound area after topical treatment with untreated, acellular fibrin glue, and either thawed (cryo) or rescue-cultured (fresh) hMSCs. (C) Single vs. triple hMSC treatment (1x vs. 3x fresh, days 0, 4, and 8). Quantification of wound area relative to initial wound area was performed using Image J. Data are shown as minimum-to-maximum boxplots, showing median values. (D, G) Comparison of wound size reduction between contralateral untreated wounds and lateral hMSCs (D) or untreated wounds (E). Control comparisons of fresh hMSCs vs. contralateral untreated and untreated vs. contralateral untreated are shown. *p≦0.05, **p≦0.01 calculated using two-way ANOVA and Tukey's multiple comparisons. Abbreviations: fresh = MSCs rescue-cultured for up to 2 days before administration to the wound; cryo = MSCs thawed immediately before application; 1× = single application on day 0; 3× = repeated applications on days d0, 4, and 8. [Figure 5] Figure 1 shows that hMSCs improve wound healing. (A) Reduction in wound size after three repeated topical treatments with either hMSCs (1 × 106 cells / cm2) in fibrin glue, acellular fibrin glue, or no treatment on days 0, 4, and 8 after wounding. Quantification of wound area relative to initial wound area was performed using Image J. *p≦0.05, ***p≦0.001 calculated using two-way ANOVA and Tukey's multiple comparisons. (B) Representative images of wounded skin after three repeated topical treatments with either hMSCs in fibrin glue, acellular fibrin glue, or no treatment, showing scab formation, especially in fibrin glue-treated wounds. [Figure 6]This figure shows that hMSCs increased lymphocyte infiltration, CD31+ angiogenesis, and CD68+ and CD163+ macrophage infiltration, tending to improve wound healing indices. Wound skin was harvested on day 14 and analyzed histologically. (A) Frequency of total cells, lymphocytes, and fibroblasts within the wound analyzed by the QuPath algorithm using HE staining. Values were normalized to the untreated contralateral site. (B) Representative images of CD31+ structures and calculated values for the total wound area, outlined in yellow. (C-E) The frequency of (C) CD31+ cells, (D) CD68+ cells, and (E) CD163+ cells relative to the total wound area was determined by immunohistochemical staining. (F-H) Histological wound healing indices: (F) epithelial thickness index (ETI) calculated by comparing epithelial thickness in uninjured skin and wound areas; (G) scar elevation index (SEI) calculated by comparing dermal thickness in uninjured skin and wound areas; and (H) collagen density after Azan staining in uninjured skin and wound areas. Quantification was performed using the QuPath algorithm. Data for individual wounds are shown. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 calculated using two-way ANOVA and Tukey's multiple comparisons. [Figure 7] Figure 1 shows that hMSCs rapidly recruit macrophages and infiltrate into the wound from the wound edge and base. (A, C, E) Immunohistochemical staining of CD68-positive macrophages and (B, D, F) CD163-positive macrophages. (A, B) Quantification as described in Figure 4. (C, D) Representative images are shown. (E, F) The wound edge is shown in yellow to highlight the histogreen-positive signal and reduce the histogreen-negative background signal to visualize the recruitment kinetics and pathways of macrophages. [Figure 8]These figures show that hMSCs are only transiently detectable in wounds. (A) Human Ku80-Histogreen staining in wound cross-sections. Left: Cross-section of the entire wound. Right: Magnification of hKu80-positive cells. On day 1, the wound edge shows fibrin glue containing hMSCs located under the intact dermis. On days 3 and 4 after hMSC application, intact hMSCs were located in the fibrin glue at the top of the wound. Although human DNA was detected in the wound by dPCR on day 9, no hKu80 signal was detected in histological sections. On day 11, very few hKu80-positive cells were observed at the base of the wound. (B) dPCR results for human DNA in rat wounds and organs. A value of 0.5 copies / μl or higher was considered a positive result. (C) hKu80 expression in frozen sections of the analyzed organs: Representative photomicrographs of samples in which human cells were detected are shown; photomicrographs of negative samples are not shown. [Figure 9] Figure 1. Summary of key findings. The present invention demonstrates a novel clinical-scale manufacturing concept consisting of six single-donor hMSC master cell banks pooled into a working cell bank from which a clinical dose of 1 x 10 hMSCs per cm of wound size, extrapolated to 70,000 cells, can be produced in just three passages. Repeated topical hMSC administration significantly accelerated wound healing in a diabetic rat model by delivering defined growth factor cargoes at specific stages of wound repair: inflammation, proliferation, and remodeling. Specifically, hMSCs mediated epidermal and dermal maturation and collagen formation, improved angiogenesis, and promoted cellular infiltration, particularly the dynamic recruitment of M2 macrophages. [Figure 10](Supplementary Figure 1) All hMSC pools exhibit similar adipogenic and osteogenic differentiation potential. hMSCs were seeded at a density of 1,000 cells / cm2 and grown to subconfluency. Adipogenic differentiation was induced using the hMSC Adipogenic Differentiation Medium BulletKit (Lonza), or osteogenic differentiation was induced using osteogenic medium (Sigma-Aldrich) composed of α-MEM / 10% FBS supplemented with 1 μM dexamethasone, 50 μM ascorbic acid, and 10 mM β-glycerophosphate. After 3 weeks under differentiation conditions, lipid vacuoles in adipogenic cultures were stained with Oil Red O, and calcium deposits in osteogenic cultures were stained with Alizarin Red S. [Figure 11] (Supplementary Figure 2): hMSCs migrate from diluted fibrin glue. Instead of applying it to a wound, hMSCs were seeded in fibrin glue onto cell culture well plates and cultured in medium supplemented with hPL or serum-free medium as a control. 4.5 hours after seeding, hMSCs migrated from the glue and achieved a typical fibroblast-like morphology. hMSCs in serum-free medium did not migrate from the gel. (Axio Vert. A1, 5x magnification). [Figure 12] (Supplementary Figure 3): Absence of systemic wound healing effects after topical hMSC application. Rats were wounded and hMSCs in diluted fibrin glue were applied topically. Comparison of wound size reduction between contralateral untreated wounds and lateral wounds treated with (A) untreated, (B) a single application of cryo-hMSCs, (C) a single application of fibrin glue, (D) a single application of fresh hMSCs, (E) repeated applications of fibrin glue, or (F) repeated applications of fresh hMSCs on days 0, 4, and 8. Wound area was quantified using Image J. **p ≤ 0.01 calculated using two-way ANOVA and Sidak's test. DETAILED DESCRIPTION OF THE INVENTION

[0018] The elements of the present invention are described below. While these elements are listed with specific embodiments, it is understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. The description should be construed as supporting and encompassing embodiments that combine two or more explicitly described embodiments, or combine one or more explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, all permutations and combinations of all elements described in this application should be considered disclosed by the description of this application, unless the context dictates otherwise.

[0019] In a first aspect, the present invention provides a method for generating a donor cell pool of expandable cells derived from bone marrow samples of multiple genetically non-identical bone marrow donors, comprising: (a) providing two or more bone marrow donor samples from genetically non-identical (or different) bone marrow donors; (b) separately passaging bone marrow-derived cells from each bone marrow donor sample of (a) into (separate) cell culture receptacles such as culture flasks or dishes (p0); (c) culturing bone marrow-derived cells from each donor separately; (d) (p1) separately subculturing a portion of each culture of bone marrow-derived cells from (c) into a new cell culture receptacle such as a culture flask or dish; (e) culturing bone marrow-derived cells from each donor separately; (f) Passaging and pooling a portion of each culture of bone marrow-derived cells from (e) into a single cell culture receptacle to obtain a donor cell pool (p2); The present invention relates to a method, including:

[0020] Method steps are performed in sequentially numbered order only to the extent technically necessary. The scope of the method claims is not to be understood as being limited by the mere arbitrary choice of method step numbering. However, preferred embodiments relate to the exact depicted order of method steps.

[0021] The present invention is based, in part, on the observation that the pooling method of the present invention structurally alters MSC preparations following passage, such that cells pooled according to the present invention produce, secrete, and possess a variety of different bioactive factors and activities. Some of these factors are illustrated in the Examples section herein below. Accordingly, specifically provided in the context of the present invention are preparations of MSCs, either cell-containing or cell-free, or cell-derived, such as lysed cells or extracts, obtained from culture of the MSC donor cell pools described hereinabove. Accordingly, provided are cellular MSC products produced according to the present invention, in vitro cellular products derived from MSCs (e.g., culture supernatants, extracellular vesicles (EVs), proteins, etc.), as well as in vivo released factors of the MSC preparations of the present invention, preparations containing factors present in MSCs, and in vivo derived factors produced from the MSCs of the present invention.

[0022] The term "non-genetically identical" refers to identity in the genetic sense, not absolute identity, which can also be expressed as "non-clonal" and refers to a genetic heritage other than that of genetically "identical" twins or inbred (non-clonal) animals that closely resemble genetic twins. This term is understood to not imply 100% identity, which would be meaningless from an epigenetic perspective or a naturally occurring single mutation. The term "monogenic," when used in the context of describing a sample or composition of cells, refers to the cells being derived from a common source or having the same (e.g., clonal) genetic background. On the other hand, the term "polygenic" refers to a composition of cells derived from different sources and having different genetic backgrounds, in the sense of not being genetically identical as defined above. In the context of the present invention, a "polygenic MSC preparation" is a composition comprising MSCs with different genetic backgrounds, for example, MSCs derived from at least two genetically distinct bone marrow donors.

[0023] The terms "passaging" or "passage" refer to the transfer of all or a portion of cells from either an initial source, such as a biological sample, or a previous cell culture into a new cell culture. Preferably, the cells are transferred to a new cell culture receptacle, such as a dish, bottle, or flask, containing fresh cell culture medium, allowing for further culturing and / or expansion. Passage may involve transferring only a specific fraction of cells, such as adherent or soluble cells. In some embodiments, passaging may involve removing non-adherent cells along with the old cell culture medium and adding new cell culture medium to the remaining adherent cells to allow further growth and expansion of the adherent cell fraction. For purposes of the present invention, a passage is counted from p0, where cells from a biological sample are transferred to a cell culture receptacle for further culturing. Subsequent passages are counted by natural numbers p1, p2, ... Pn. A passage is defined as the time from the completion of the first transfer to the moment the next transfer begins. Essentially, a new passage begins as soon as a new receptacle or medium is provided.

[0024] In the present invention, a cell culture passage line of a monogenic cell preparation begins at p0 and remains monogenic until the first pooling occurs. In the method of the present invention, it is preferred that at least p0 and p1 are monogenic, with the cell cultures being combined (pooled) at the beginning of p2. Cultivation of multiple monogenic cell cultures in parallel or sequentially (with some of the cells cryopreserved and thawed for pooling) is also encompassed by the present invention.

[0025] "Cell culture receptacle" shall be understood to refer to any container suitable for cell culture, preferably for growing and expanding cells in or on a medium. Typical cell culture receptacles are flasks, plates, multi-well plates, bioreactors, etc.

[0026] As used herein, the term "bone marrow-derived cells" (BMDCs) refers to a population of cells derived from a bone marrow sample that includes or comprises mesenchymal stem (or stromal) cells (MSCs).

[0027] In one embodiment, the method of the present invention further comprises, following (f), the step (f') of cryopreserving the donor cell pool as a working cell bank.

[0028] In another embodiment, the method of the present invention further comprises a step (g) of culturing the donor cell pool, either directly from step (f) or after thawing a cell sample of the working cell bank of the above embodiment.

[0029] The term "cellular material," as used in the context of the present invention, specifically refers to biological cellular material, e.g., material comprising live or dead cells, preferably live cells, as well as any secreted factors derived from the culture of such cells. However, in certain preferred embodiments, the present invention relates to cellular material comprising live biological cells. The term "biological cells," in the context of the present invention, preferably refers to mammalian cells, most preferably human cells. Certain embodiments of the present invention do not include mesenchymal stromal cells, but relate to acellular compositions derived from such cells, e.g., from such cells. In the present disclosure, the term "mesenchymal stromal cells" refers to multipotent stromal cells that can differentiate into various cell types, including, but not limited to, osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells), and adipocytes (fat cells). These cells are also known as "mesenchymal stem cells" due to their multipotency. This biologically important cell population is capable of supporting hematopoiesis, can differentiate in vitro into mesenchymal and non-mesenchymal lineages, can suppress allogeneic responses, and appears to be non-immunogenic.

[0030] In some embodiments, MSCs according to the present invention are provided as monogenic compositions, and thus such compositions contain cells of only one genetic background (or are derived from a single donor subject). However, preferred embodiments of the present invention relate to cell preparations containing BMDCs derived from multiple genetically distinct donor subjects. In the pooling method of the present invention, multiple monogenic pools of BMDCs are pooled into one cell culture at a specific passage, so that the BMDC preparation can change genetic background from monogenic to polygenic. The number of donors providing the sample to be pooled for subsequent BMDC isolation is preferably two or more, preferably from at least 3, 4, 5, 6, 7, 8, 9, or 10 or more genetically distinct donor subjects. Preferably, BMDCs according to the present invention are obtained from a bone marrow sample, such as a pooled bone marrow mononuclear cell (BMNC) fraction grown at passage p2. In the context of the method of the present invention, it is preferred to transfer only adherent cells to a new passage.

[0031] As used herein, the term "master cell bank" or "MCB" refers to a culture of cells grown from a single donor sample, i.e., monogenic, aliquoted into storage containers, and preferably stored under cryopreservation conditions known to those of skill in the art. These cells may be suitable for subsequent use in the production pooling cultures of the present invention, ultimately yielding a therapeutic clinical product. The term "working cell bank" or "WCB" may refer to a single donor bank of multiple monogenic cell cultures, or, preferably, to an already pooled polygenic culture of cells pooled and optionally grown from cultures derived from a single donor sample, aliquoted into storage containers, and preferably stored under cryopreservation conditions known to those of skill in the art. In certain embodiments, the method of the present invention involves transferring a portion of the cells between p0 and p1 into a storage receptacle and freezing to obtain a donor cell master cell bank, and thawing and passage of the cells from the storage receptacle into cell culture receptacles for further cell culture in step (c) to continue p1.

[0032] In some embodiments, the method of the present invention further comprises a step (h) (p3) of passaging a portion of the pooled cells cultured in step (g) into (separate) cell culture receptacles. Preferably, the cells obtained from the cultured cells in P3 can be used as a clinical product suitable for medical use.

[0033] The method of the present invention is preferably for the isolation and generation of a pool of mesenchymal stromal cells (MSCs).

[0034] In the context of the present invention, any subject, patient or donor is preferably a mammal, most preferably a human.

[0035] The method of the present invention may further include an optional additional step of testing cells at any passage, cell bank, or clinical product. Testing methods may include testing for activity and cell number. Detailed testing is described in the Examples section below. In either case, testing may include determining the presence of one or more growth factors in the cells or cell culture medium. Such growth factors may be selected from the group consisting of brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), granulocyte-colony stimulating factor (G-CSF), hepatocyte growth factor (HGF), IL-1α, IL-6, LIF, osteopontin, vascular endothelial growth factor (VEGF)-A, fibroblast growth factor (FGF-2), transforming growth factor (TGF)-β, prostaglandin E (PG) E-2, and indoleamine 2,3-dioxygenase (IDO) after priming. Any one or any combination of the above may be tested to assess the quality of the cells or cell preparation.

[0036] In a second aspect, the present invention relates to working cell banks and / or clinical products produced by the methods of the present invention.

[0037] In a third aspect, the present invention relates to a preparation of clinical-grade pooled mesenchymal stromal cells (MSCs) produced by the method of the present invention.

[0038] In a fourth aspect, the present invention relates to a preparation of clinical-grade pooled mesenchymal stromal cells (MSCs) for use in treating a disease or condition in a subject.

[0039] In a fifth aspect, the present invention relates to a method for producing a therapeutically active composition, such as a composition containing cell-free growth factors, comprising carrying out a method according to the invention or providing a working cell bank and / or clinical product or preparation of any aspect of the invention, followed by culturing and / or expanding MSC material in cell culture and harvesting the composition in which the MSC material has secreted one or more cellular factors, to obtain the therapeutically active composition. Such a composition may preferably comprise any vesicles, such as ECV, derived from a cell preparation of the invention, and / or growth factors as described.

[0040] It may be preferred that the therapeutically active composition is essentially cell-free, such as cell-conditioned culture medium, optionally after removing cultured cells, e.g., by centrifugation. Preferably, the medium is enriched with MSCs of the invention.

[0041] It is further preferred that the therapeutically active composition comprises one or more growth factors, preferably selected from brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), granulocyte colony-stimulating factor (G-CSF), hepatocyte growth factor (HGF), IL-1α, IL-6, LIF, osteopontin, vascular endothelial growth factor (VEGF)-A, fibroblast growth factor (FGF-2), transforming growth factor (TGF)-β, prostaglandin E (PG) E-2, and indoleamine 2,3-dioxygenase (IDO) after priming.

[0042] In a sixth aspect, the present invention relates to a therapeutically active composition containing one or more growth factors made by the method of the present invention.

[0043] In a seventh aspect, the present invention provides a pharmaceutical composition comprising a cell or cell-free material of any aspect of the invention or obtained or obtainable by any aspect of the invention.

[0044] The methods and compositions described herein are useful for pharmaceutical applications, such as therapeutics. Thus, for example, mesenchymal stromal cells can be administered to animals to treat blocked arteries, including those in the extremities (i.e., arms, legs, hands, and feet), as well as in the neck or various organs. For example, mesenchymal stromal cells can be used to treat blocked arteries supplying the brain and treat or prevent stroke. Mesenchymal stromal cells can also be used to treat blood vessels in the embryonic and postnatal cornea and to provide glomerular structuring. In another embodiment, mesenchymal stromal cells can be used to treat both internal and external wounds, as well as dermal ulcers found on the feet, hands, legs, or arms, including, but not limited to, dermal ulcers resulting from diseases such as diabetes and sickle cell anemia. Furthermore, mesenchymal stromal cells or their acellular derivatives can be administered intranasally. MSCs and / or their acellular derivatives can be used as immunomodulatory therapeutics for autoimmune diseases, alloimmune conditions (including, but not limited to, graft-versus-host disease), and antitumor therapy. Any of the uses described herein may involve the use of cellular material or any cell-free conditioned composition produced according to the present invention.

[0045] Furthermore, because angiogenesis is involved in embryonic implantation and placental formation, mesenchymal stromal cells can be used to promote embryonic implantation and prevent miscarriage. Additionally, mesenchymal stromal cells can be administered to fetal subjects, including humans, to promote the development of the vasculature in the fetal subject. In another embodiment, mesenchymal stromal cells can be administered to a newborn or prenatal subject to promote cartilage resorption and bone formation, as well as promote proper growth plate morphogenesis. Mesenchymal stromal cells can be genetically engineered with one or more (poly)nucleotides encoding therapeutic agents or to further improve the function of mesenchymal stromal cells. Genetic engineering can also include genome editing of mesenchymal stromal cells, for example, using designer nucleases. Polynucleotides can be delivered directly to mesenchymal stromal cells or via an appropriate expression vehicle. Expression vehicles that can be used to genetically engineer mesenchymal stromal cells include, but are not limited to, retroviral vectors, adenoviral vectors, and adeno-associated viral vectors. The MSCs of the present invention can be genetically engineered, for example, to overexpress TERT, thereby immortalizing the cells. The MSC preparation or mesenchymal stromal cells of the present invention can also be used for stem cell transplantation. There is further provided the use of the MSC preparation or MSCs of the present invention in the production of a bone replacement material.

[0046] As used herein, the terms "of the invention," "in accordance with the invention," "according to the invention," and the like are intended to refer to all aspects and embodiments of the invention described and / or claimed herein.

[0047] As used herein, the term "comprising" is to be interpreted as encompassing both "including" and "consisting of," with both meanings specifically intended, and thus referring to individually disclosed embodiments according to the present invention. As used herein, "and / or" shall be interpreted as a specific disclosure of each of the two specified features or components, regardless of the presence or absence of the other. For example, "A and / or B" shall be taken as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if individually defined herein. In the context of the present invention, the terms "about" and "approximately" indicate an interval of precision understood by a person skilled in the art to still ensure the technical effect of the feature in question. This term typically indicates a deviation from the specified numerical value of ±20%, ±15%, ±10%, and, for example, ±5%. As will be understood by a person skilled in the art, the specific deviation regarding the numerical value for a given technical effect depends on the nature of the technical effect. For example, natural or biological technical effects may generally have greater such deviations than man-made or engineered technical effects. As will be understood by one skilled in the art, the specific such deviations regarding the numerical value for a given technical effect will depend on the nature of the technical effect. For example, natural or biological technical effects may generally have greater such deviations than man-made or engineered technical effects. When an indefinite or definite article is used when referring to a singular noun, such as "a," "an," or "the," this includes the plural of that noun unless specifically stated otherwise.

[0048] It will be understood that adapting the teachings of the present invention to a particular problem or environment, and incorporating variations of the present invention or additional features thereto (such as further aspects and embodiments), is within the skill of one of ordinary skill in the art in light of the teachings contained herein.

[0049] Unless otherwise dictated by context, the feature descriptions and definitions presented above are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0050] All references, patents, and publications cited herein are hereby incorporated by reference in their entirety. [Example]

[0051] Certain aspects and embodiments of the present invention will now be described, by way of example, with reference to the description, figures, and tables set forth herein. Such examples of methods, uses, and other aspects of the present invention are merely representative and should not be construed as limiting the scope of the present invention to only such representative examples.

[0052] The examples show:

[0053] Example 1: Closed system and pooled hPL allows for scaled-up production of pooled hMSC doses with defined trophic factor content For scale-up and GMP manufacturing, hMSCs were cultured in standard Nunclon™ Delta flasks (175 cm per flask). 2 ) and CELLSTACK™ (636 cm per stack) with a larger culture surface that allows seeding, medium exchange, and harvesting in a closed MC3 system. 2 ) and hMSCs were cultured simultaneously. Growth kinetics and hMSC surface marker expression were identical (not shown). Due to optimization of medium exchange and passaging / harvesting procedures, especially due to the reduction of hands-on time in cell culture, production of clinical-scale doses was feasible in the closed MC3 system.

[0054] Compared with hMSCs from Pool 1 (approximately 6,000 extrapolated doses) and Pool 3 (approximately 50,000 extrapolated doses), hMSCs from Pool 2 achieved the maximum extrapolated cell number that could be produced to match the highest target cell dose (approximately 70,000 extrapolated doses) (Figures 1A, 1B, 3A, and 3B). Expression of binary (absent or present) MSC markers was identical for all hMSC pools, consistent with the guidelines established by the International Society for Cellular Therapy (Figure 3C). Functional characterization of the hMSC pools demonstrated similar adipogenic and osteogenic differentiation potential (Supplementary Figure 1) and immunomodulatory potential measured by inhibition of PHA-driven T cell proliferation (Figure 3D). Live cell imaging also demonstrated no differences between the hMSC pools in proliferation, scratch wound healing, vascular tube formation support, and chemotactic migration (Figures 3E–H). However, day-to-day and operator-to-operator variability was evident in the latter assay, highlighting the need for better assay standardization.

[0055] Because hMSCs from Pool 2 achieved the highest calculated extrapolated clinical dose with similar properties compared to hMSCs from Pool 1 and Pool 3, we selected hMSCs from Pool 2 for further preclinical evaluation.

[0056] The delivery of trophic factors is an important MoA of hMSCs (13). Therefore, we quantitatively evaluated candidate trophic factors for wound healing. Notably, we analyzed hMSC lysates, which reflect actual clinical products, rather than simply cell culture supernatants collected during expansion. Specifically, we detected BDNF, EGF, G-CSF, HGF, IL-1α, IL-6, LIF, osteopontin, VEGF-A, FGF-2, TGF-β, PGE-2, and inducible IDO-1 in hMSCs and calculated their contents per applied hMSC dose (Table 1). GM-CSF, IL-1β, NGF-β, angiopoietin, IFN-γ, IL-2, and TNF-α were below the detection limit of the assay. These growth factors are active at different stages of wound healing.

[0057] [Table 1] TIFF2025526873000002.tif87170

[0058] Considering that medium supplements influence the final trophic factor composition of hMSC lysates (41), we also tested the hPL batch used in this study (Table 2). Here, we detected high concentrations of TGF-β1, EGF, PDGF-AB, and VEGF-A, reflected by relatively high amounts of TGF-β1 and VEGF-A in the hMSC dose (Table 1).

[0059] [Table 2]

[0060] Example 2: hMSCs migrate from fibrin glue and improve skin wound healing in diabetic rats For cell application, we used 1:10 diluted TISSEEL fibrin glue as a cell carrier and followed the protocol established by Yufit et al. (9). In a pilot in vitro experiment, we verified that hMSCs formulated in 1:10 diluted fibrin glue could migrate out of the glue. The diluted fibrin glue required approximately 7 min to polymerize into a gel. After 4.5 h, hMSCs began to migrate out of the glue and into the culture vessel, and hMSC migration increased over time (Supplementary Figure 2). Notably, migration was not induced in serum-free conditions, demonstrating targeted hMSC migration.

[0061] To evaluate the wound-healing potential of pooled hMSCs in vivo, three pilot studies were conducted in preparation for this study. In each study, two 8-mm-diameter circular wounds were created per animal and were either left untreated, treated with acellular fibrin glue, or treated with hMSC-infused fibrin glue (Figures 2A and 2B).

[0062] In pilot study 1, we compared freshly thawed hMSCs (cryo) with rescue-cultured hMSCs (fresh) to assess the ultimate cryoinjury. From day 4 onward, wounds treated with fresh hMSCs healed slightly better than hMSCs (cryo) (Figure 4B). At day 12, wound sizes were significantly smaller in hMSCs (cryo)-treated wounds compared with untreated wounds. Acellular fibrin glue itself accelerated wound healing compared with untreated wounds, but slightly delayed it compared with hMSC-treated wounds (d10 and d12, Figure 4B). Based on these data, we concluded that hMSCs exhibit minimal cryoinjury and supported the use of rescue-cultured fresh hMSCs in subsequent experiments.

[0063] In pilot study 2, we evaluated whether wound healing could be further accelerated by repeatedly applying fresh hMSC doses on days 0, 4, and 8. Given that hMSC treatments contain a wide variety of growth factors known to be required for activity during the inflammatory, proliferative, and remodeling phases of wound healing, we applied hMSCs on days 0, 4, and 8, respectively, reflecting different wound healing stages (Figure 2A). Starting on day 4, wounds treated with hMSCs tended to be smaller than control-treated wounds, but from day 8 onward, wounds treated three times with hMSCs were significantly smaller than controls (Figures 4A and 4C).

[0064] Example 3: Topically applied hMSCs do not exert a systemic wound healing effect Furthermore, blood samples collected during the pilot study were analyzed comparing the numbers of white blood cells (WBCs), neutrophils, lymphocytes, and platelets on days 0 and 14. In the control setting, all blood cell counts appeared to increase on day 14. However, 9 of 15 hMSC-treated animals showed a decrease in the number of WBCs, especially lymphocytes, compared to day 0. This effect became more pronounced after repeated hMSC application (not shown). None of the control animals showed this trend.

[0065] Therefore, in pilot study 3, we asked whether hMSCs could exert a systemic effect and affect the healing of contralateral wounds to which hMSCs were not applied locally. Statistical analysis revealed that only wounds treated locally with three doses of hMSCs at different time points significantly improved healing. Contralateral untreated sites showed healing comparable to that of untreated wounds (Figures 4D and 4E, Supplementary Figure 3). These data suggest that locally applied hMSCs exert their therapeutic wound healing effects only locally.

[0066] Based on the results of these pilot studies and power-based sample size calculations, we designed the present study to (i) use fresh rescue-cultured hMSCs, (ii) apply repeated hMSC doses on days 0, 4, and 8, and (iii) reduce the number of control wounds according to the 6R principle to exclude systemic effects.

[0067] The results of this study supported the significant wound healing effects of hMSCs. Wounds treated with three consecutive hMSC doses were significantly smaller than both controls on days 10, 12, and 14 (Figures 5A and 5B). Importantly, some of the hMSC-treated wounds had already closed 12 days after wound initiation. In both control groups, the initial wounds were not completely healed until day 14. We found that both untreated and acellular fibrin glue-treated control wounds exhibited similar wound healing rates. In these series of experiments, we observed extensive scab formation in fibrin glue-treated wounds but not in hMSC-fibrin glue-treated wounds (Figure 5).

[0068] Data from the pilot and current studies demonstrated that hMSCs significantly improved wound healing compared with both control groups, but did not confirm the initially observed trend of reduced circulating lymphocytes in the peripheral blood after topical hMSC application.

[0069] Example 4: hMSCs increase CD31-positive capillaries and recruit CD68- and CD163-positive macrophages to healing wounds Because we observed accelerated wound healing in hMSC-treated wounds, we performed histological analysis. To gain insight into cellular infiltration into the wound, we identified total cells, lymphocytes, and fibroblasts based on their typical nuclear and cellular phenotypic characteristics (Figure 6A). Although we detected more lymphocytes in hMSC-treated wounds than in untreated and fibrin glue-treated wounds, the number of fibroblasts and total cells did not appear to be affected by hMSC treatment (Figure 6A).

[0070] In the next step, immunohistochemical staining was performed for CD31, which indicates tissue angiogenesis; CD68, a pan-macrophage marker; and CD163, a marker of M2 subtype anti-inflammatory macrophages activated during wound healing in mice, which promote anti-inflammatory functions, extracellular matrix formation, and angiogenesis (42). We found that wounds repeatedly treated with hMSC doses had an increased number of CD31+ capillaries compared with untreated and fibrin glue-treated controls (Figures 6B and 6C). hMSC-treated wounds also showed higher percentages of CD68+ and CD163+ infiltrating macrophages compared with both control groups (Figures 6D and 6E). Detailed microscopic wound assessment at different time points after wound initiation (part of the biodistribution study) revealed that macrophages gradually infiltrated from the wound edge (d3), followed by the wound base (d9), and finally progressing to the wound tip tissue by d11 (Figure 7). In hMSC-treated wounds, CD68+ cell infiltration expanded toward the apical layer compared with both controls (Figures 7C and 7E). This infiltration accelerated in hMSC-treated wounds on day 9 and then declined to levels similar to those in both controls by day 11 (Figure 7A). From day 4, hMSCs promoted the infiltration / differentiation of CD163-expressing macrophages, whereas neither control-treated wound showed an increase in CD163-positive cells. Only in hMSC-treated wounds did the CD163+ signal peak at day 9 (Figure 7B), with positive signals extending throughout the entire wound area and into the apical layer (Figures 7D and 7F).

[0071] Example 5: hMSCs improve epithelial thickness, reduce scar elevation, and increase collagen density in healing wounds Having demonstrated that hMSCs increased angiogenesis and induced not only CD68-positive but also CD163-positive macrophage infiltration, we sought to gain further insight into the dynamics of wound healing. Here, we used a histological scoring system (39). First, we calculated the epithelial thickness index (Figure 6F). Nearly all wounds showed epithelial thickening, indicating the healing stage (39). Although not statistically significant, the mean values suggested that hMSC-treated wounds showed the thinnest epithelial thickness, followed by fibrin and then untreated wounds (Figure 6F). Second, we calculated the scar elevation index (SCRI). Although all wounds showed dermal hypoplasia, hMSC-treated wounds were already close to normal and significantly different from untreated wounds, indicating that they were already better developed wounds compared with both controls (Figure 6G). Third, we calculated collagen density based on the intensity of blue Azan staining and compared it to the respective unwounded dermis. After migrating into the wound, fibroblasts gradually produce ECM and collagen fibers. During wound healing, especially during the proliferation phase, collagen accumulates in the wound, resulting in a darker blue Azan stain. Our results showed significantly higher collagen deposition and density in hMSC-treated wounds compared with controls, indicating improved collagen deposition, on day 14 of the experiment (Figure 6H).

[0072] Example 6: hMSCs are only transiently detectable in wounds To assess the fate and biodistribution of locally applied hMSCs within the wound and in distant organs over time, animals were sacrificed on days 1, 2, 3, 4, 9, and 11, followed by histological and dPCR-based quantification of human cells.

[0073] By staining wound sections for human nuclear Ku80 expression (38), we identified locally applied hMSCs in areas of fibrin glue scattered within the nonhuman tissue on days 3 and 4, whereas hMSCs were undetectable at later time points (Figure 8A).

[0074] Thus, we confirmed the presence of human DNA in hMSC-treated wounds. Levels decreased over time, suggesting that hMSCs were gradually eliminated from the wounds (Figure 8B). Interestingly, traces of human DNA were also detected in acellular fibrin glue-treated wounds, but never in untreated wounds, suggesting that fibrin glue may contain low levels of human DNA. Human DNA was also detected in the livers of hMSC-treated rats on days 1, 2, 4, and 11. Histological cross-checking revealed that the hKu80 signal was located in the cytoplasm, not in the nuclei, of rat hepatocytes (Figure 8C).

[0075] We also detected traces of human DNA in the livers of rats whose one wound was treated with fibrin (Figure 8B). These results were confirmed by histological analysis. Again, hKu80 staining was cytoplasmic (Figure 8C). It appears that hMSCs, as well as fibrin glue fragments, were transported from the wound site to the liver and phagocytosed by hepatocytes, although to a lesser extent. Traces of human DNA were also found in the spleens of hMSC-treated rats on days 1, 4, and 11, and in the spleens of animals with fibrin glue-treated wounds on day 1. Histological analysis confirmed this result (Figure 8C). The presence of human DNA in the lungs of hMSC-treated rats was detected by dPCR on days 1 and 2. No human DNA was found in the lungs of fibrin glue-treated rats.

[0076] Methods and Materials BM-derived hMSCs: isolation, culture and characterization Human BM-MNCs were obtained by puncturing the iliac crest of healthy BM donors (ethical vote no. 329 / 10, Ethics Committee, University Hospital Frankfurt am Main, Germany). hBM-MNCs were cultured in Nunclon™ Delta flasks containing glutamine (Lonza, Cologne, Germany), 6% pooled virus-inactivated human platelet lysate (hPL). 26(MultiPL'100i, Macopharma, Tourcoing, France), 100,000 cells / cm in 93% α-MEM containing 1% penicillin / streptomycin (Thermo Fisher Scientific, Darmstadt, Germany) and 2 IU heparin (Ratiopharm GmbH, Ulm, Germany). 2 After 24 hours, non-adherent cells were removed by rinsing with PBS (Thermo Fisher Scientific) and changing the culture medium, and hMSCs were grown from the adherent cell fraction (15). For scale-up and GMP-compliant manufacturing, hMSCs were also cultured in a CellStack with a larger culture surface, which allows seeding, medium change, and harvesting in a closed system (MC3 system, Macopharma). After reaching subconfluence, hMSCs were split using TrypLE™ Select (Thermo Fisher Scientific) to a density of 1000 cells / cm. 2 hMSCs were seeded at a density of 1 × 10 or cryopreserved as single-donor MCBs in α-MEM, 33% hPL, 5% DMSO (Sigma-Aldrich, Taufkirchen, Germany). To equalize for individual differences, single-donor hMSCs from six randomly selected donors were then thawed and cultured at equal cell numbers (e.g., 1 × 10) at the start of either passage 1, passage 2, or passage 3. 6 hMSCs were pooled six times (pool 1, pool 2, and pool 3, respectively). At the end of passage 3, hMSCs were cryopreserved as the final ("clinical") product. Pool 1 and pool 2 were designated as pooled WCB (Figure 1A). hMSCs were confirmed to be mycoplasma-free (Venor™ GeM Classic, Minerva Biolabs GmbH, Berlin, Germany) and endotoxin-free (Endosafe™ nexgen-PTS™, Charles River Laboratories, Freiburg, Germany).

[0077] The population doubling number is calculated using the formula: Population doubling number (PD) = (ln(harvesting / seeding)) / ln2 The maximum achievable cell number is calculated using Maximum achievable cell number = input amount x 2 (集団倍加数) It was calculated by:

[0078] Maximum achievable cell number and target cell dose equivalent (wound size 1 cm) at the end of passage 3 2 1x10 6 hMSCs) were extrapolated for pools 1 to 3, respectively (32).

[0079] hMSCs were characterized using a series of in vitro test systems: First, marker expression (binary markers, either absent or present in hMSCs (33)) was assessed by flow cytometry (32). Second, adipogenic differentiation was induced using the hMSC Adipogenic Differentiation Medium BulletKit™ (Lonza, Basel, Switzerland), and osteogenic differentiation was induced using osteogenic medium (Sigma-Aldrich) composed of α-MEM, 10% FBS, supplemented with 1 μM dexamethasone, 50 μM ascorbic acid, and 10 mM β-glycerophosphate. After 3 weeks of differentiation, cells were stained lineage-specifically. Lipid vacuoles in adipogenically differentiated cultures were stained with Oil Red O, and calcium deposits in osteogenically differentiated cells were stained with Alizarin Red. Third, the ability of hMSCs to inhibit T cell proliferation in vitro was assessed (32). Briefly, hMSCs were pre-seeded, and pooled peripheral blood mononuclear cells (PBMNCs) labeled with CellTrace™ Violet (Thermo Fisher) were added and further stimulated with phytohemagglutinin-L (PHA-L, 10 μg / mL, Sigma-Aldrich) or maintained as unstimulated controls. Proliferation of PBMNCs was assessed using flow cytometry after 5 days, and hMSC-mediated inhibition was calculated. Fourth, live cell imaging was performed using an Incucyte™ Zoom instrument (Sartorius AG, Hertfordshire, UK) and analyzed using the Incucyte™ analysis algorithm. First, 1 cm 2hMSC proliferation was assessed by seeding 200 hMSCs per 1000 mm cell culture medium and monitoring the increase in cell confluence over time. The 96-hour time point was chosen to compare pools 1 to 3. Second, a scratch wound healing assay of the hMSC monolayer was performed. Specifically, hMSCs were seeded at 60,000 cells / cm. 2 hMSCs were seeded at 1000 x g / cm² and incubated overnight. A wound scratch was then created using a 96-pin Incucyte™ woundmaker tool. Wound closure (wound density at different time points relative to the initial wound size) was calculated over time, and the value at 24 h was used for comparison. Third, angiogenic tubular network formation in hMSC monolayers was assessed as previously described (34). hMSCs were seeded at 60,000 cells / cm². 2 After 60 minutes, 15,000 green fluorescent protein (GFP)-positive human umbilical vein endothelial cells (HUVECs) were added. Human adipose-derived stromal cells (hASCs) served as a positive control and were used for normalization of individual experiments. Network length (mm / mm 2 ) was chosen as a parameter for quantitative analysis. Fourth, the chemotactic migration of hMSCs was assessed (35). Briefly, fibronectin was coated onto the insert plate of an Incucyte™ ClearView 96-well plate. Subsequently, 1000 hMSCs were seeded, and the plate was mated with a reservoir plate containing serum-free medium or hPL-containing medium. hMSC migration was monitored for 48 hours and analyzed as "number normalized to the initial peak value."

[0080] The trophic factors in hMSC lysates were quantified using Luminex and ELISA techniques (32). Briefly, 1 × 10 6 pieces~10×10 6hMSCs were harvested. The hMSC pellet was lysed in ice-cold ProcartaPlex™ cell lysis buffer, centrifuged at maximum speed, and the supernatant was stored at -80°C until assayed. Transforming growth factor-β1 (TGF-β1) and prostaglandin E2 (PGE2) were analyzed by ELISA (Biorbyt Ltd., Cambridge, UK, and Cayman Chemical, Ann Arbor, MI, USA, respectively). Fibroblast growth factor 2 (FGF2) was analyzed by singleplex assay, and all other trophic factors were analyzed using a ProcartaPlex™ custom multiplex panel (Thermo Fisher Scientific).

[0081] hMSC IDO-1 production was stimulated with 20 ng / mL tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interferon-γ (IFN-γ) for 48 hours. Subsequently, hMSCs were harvested and counted. The pellets were lysed (300 mM NaCl, 50 mM Tris, 2 mM MgCl2, 0.05% NP40, 1× protease / phosphatase inhibitors) and centrifuged at maximum speed. The supernatants were stored at -80°C until ELISA (Cloud-Clone Corp., Katy, TX, USA) was performed.

[0082] To assess the trophic factor content in the pooled hPL batch (MultiPL'100i; batch number 11219267DM) used in this study, two different bags were tested by ELISA (Bio-techne; FGF (#SFB50), vascular endothelial growth factor-A (VEGF-A; #SVE00), epidermal growth factor (EGF; #SEG00), platelet-derived growth factor-AB (PDGF-AB) (#SHD00C), insulin-like growth factor-1 (IGF-1) (#SG100), and TGF-β1 (#SB100B)).

[0083] Wound healing model Animal experiments were approved by the local ethical committee (G142-19, Regierungspraesidium Karlsruhe, Germany). Zucker diabetic rats were chosen as a model of impaired wound healing (31). A total of 66 6-week-old male rats (ZDF (obese fa / fa), ZDF-Leprfa / Crl; Charles River Laboratories, Châtillon, France) were used. After arrival, rats were housed in groups of two and fed a special high-fat diet (Purina #5008, ssniff Spezialdiaet GmbH, Soest, Germany) ad libitum for 6 weeks to induce type II diabetes (Figure 2A). Rats were weighed every 2 days, and nonfasting blood glucose was measured weekly (Accu-Chek™ Aviva, Roche Diabetes Care, Mannheim, Germany). Typically, animals were considered diabetic when they reached a glucose level of 300 mg / dL 3 weeks after starting the diet. Only diabetic rats were used in the wound healing experiments. Rats with blood glucose levels above 600 mg / dL were fed a normal diet until their blood glucose levels decreased.

[0084] At 12 weeks of age, rats were anesthetized with isoflurane (CP-Pharma 1 mL / mL, induction with 5% isoflurane + oxygen 5 L / min and maintenance with 2%-3% isoflurane + oxygen 1 L / min), and 0.8 mL of blood was collected. For wound preparation, the rat's back was shaved, the surgical field was disinfected, and two wounds were created with an 8 mm skin biopsy punch (WDT™, Garbsen, Germany) 1.5 cm posterior to the scapula and 1.5 cm bilaterally from the spine. Only the skin was removed, leaving the skeletal muscle fascia intact. Depending on the experimental setup, control animals were left completely untreated, or one wound was left untreated and the other was treated with acellular fibrin glue. In other animals, one wound was treated with fibrin glue + hMSCs, and the contralateral wound served as a control, either left untreated or treated with acellular fibrin glue (Figure 2B). After surgery, 10 mL of saline was injected subcutaneously to prevent dehydration and speed up post-anesthesia recovery. A wound dressing was applied (Curapor™, Lohmann-Rauscher, Rengsdorf, Germany) to protect the wound from contamination or cutting. This dressing was changed every other day. 200 mg / kg of metamizole sodium was used for analgesia (Novaminsulfon™ injection 500 mg / mL, Bela-pharm, Vechta, Germany) and given by subcutaneous injection for 4 days.

[0085] For topical application of cells, a commercially available fibrin sealant syringe system was used (TISSEEL, Baxter Deutschland GmbH, Unterschleissheim, Germany). hMSCs were either thawed (cryo) or trypsinized after a brief rescue culture (fresh; cells were thawed and cultured for up to 2 days to recover from terminal cryo-damage and restart metabolism), washed, counted, and placed in 50 μL of pre-diluted fibrinogen / aprotinin solution (final concentration 5 mg / mL) at 5 × 10 5The fibrin glue was formulated at a density of 1000 viable hMSCs (9). Immediately prior to application to the wound, the cell suspension was drawn into one syringe of the Duplojet device, and the other syringe contained prediluted thrombin solution (final concentration 25 mg / mL). Both components were combined using the TISSEEL Duplojet system to formulate the fibrin glue. Then, for each wound, 50 μL of fibrinogen containing hMSCs and 50 μL of thrombin were applied onto the wound, covering the entire wound 1 cm. 2 1x10 6 A dose of hMSCs was obtained. The glue was allowed to polymerize in air for 7 minutes before applying the wound dressing.

[0086] To assess the ability of hMSCs to migrate from the gel, an in vitro migration assay was performed. Briefly, fibrin glue containing hMSCs was applied to the wells of a 24-well plate. Subsequently, migration of hMSCs into culture medium supplemented with hPL as an attractant or serum-free medium as a control was assessed microscopically.

[0087] Following the 6R principle, we conducted three pilot studies with three to five animals each. First, to pretest the ultimate cryopreservation injury, we compared freshly thawed (cryo) hMSCs with freshly thawed rescue-cultured hMSCs (28-30). Second, we compared a single injection (d0) with repeated hMSC administration (days 0, 4, and 8) to apply hMSCs during the inflammatory, proliferative, and remodeling phases (Figure 2A). Notably, despite the smaller wound size, we applied the same cell dose as on day 0. Third, we investigated the ultimate systemic effects of hMSCs. Here, one rat was assigned to one group in which the contralateral site was treated with hMSCs, while the contralateral site served as a control, and compared with a group of animals with only a control-treated wound.

[0088] Using the results from the pilot study, a power analysis was performed to calculate the number of animals for the main study, in which culture-adapted fresh hMSCs were applied repeatedly, but the treatment of the two wounds was randomly selected (n = 42 rats in total in the present study). To overcome potential breed-specific bias, the experiment was performed in different experimental cohorts.

[0089] Additionally, a biodistribution study was performed in which animals were sacrificed on days 1, 2, 3, 4, 9, and 11 (one animal per group), where both wounds were either control (untreated / fibrin group) or treated with hMSCs. The wounds, liver, spleen, and lungs were harvested, snap-frozen, and analyzed for the presence of human cells by immunohistochemical staining and digital PCR (dPCR).

[0090] For each animal, the wound was measured and photographed orthogonally at every other day's wound dressing change. Wound area was measured using ImageJ (36). In addition, blood samples were collected and blood counts were performed (CELL-DYN Ruby, Abbott GmbH, Wiesbaden, Germany) before the animals were sacrificed 14 days later. First, rats were fully anesthetized with isoflurane and then intracardially injected with 100 mg / kg ketamine and 5 mg / kg xylazine. To avoid autolysis, the wound and organs were immediately removed. Wounds were cut in half for full wound center analysis, fixed in paraformaldehyde (PFA) and embedded in paraffin, or snap-frozen in Tissue-Tek™ and cryomold.

[0091] Histological and immunohistological analysis Standard hematoxylin-eosin (HE) and Azan staining was performed on 5 μm-thick sections after the organs were fixed in 4% PFA and embedded in paraffin.

[0092] To investigate whether hMSCs promote host cell infiltration into wounds, we used the QuPath algorithm (37), based on artificial intelligence and a random tree classifier, to analyze HE staining. First, the wound was defined as a region of interest. Second, automated cell detection was performed to determine the total cell number in this region. Using the QuPath-based cell classification, fibroblasts and lymphocytes were distinguished based on nuclear staining (uniform and intense in lymphocytes compared to fibroblasts) and cell morphology (elongated fibroblasts versus round lymphocytes). In addition, a "combined classifier" was used to improve the identification of lymphocytes, which are characterized by a significantly more pronounced circularity compared to fibroblasts.

[0093] Heidenhain azantrichrome staining was performed to assess collagen fiber deposition. The mean blue intensity was taken as a measure of collagen density and dermal maturity. For this purpose, the "intensity mean value: blue" function was used (Zeiss Zen 3.0 blue edition, Carl Zeiss Microscopy, Oberkochen, Germany). This tool calculates the mean intensity (pixel value) of a selected region of interest. A darker blue color reflecting higher collagen density has a lower pixel value than the lighter blue staining of wounds with fewer collagen fibers. The pixel value of the wound tissue was compared to that of the surrounding undamaged dermis, with lower intensity corresponding to more collagen deposited in the granulation tissue. Mean blue intensity (%) = mean blue intensity value of wound × 100 / mean blue intensity value of intact dermis

[0094] Immunohistochemical staining was performed to assess the degree of angiogenesis (CD31+ endothelial cells), immune cell infiltration (CD68+ and CD163+ macrophages), and the presence of transplanted hMSCs (human Ku80+ cells (38)). Frozen sections (10 μm) were fixed with 4% PFA for 10 min. Nonspecific binding sites were blocked with 1% bovine serum albumin (BSA, PAN-Biotech, Aidenbach, Germany), 0.2% fish skin gelatin (Sigma-Aldrich), and 0.1% Triton X (Carl Roth, Karlsruhe, Germany) in Tris-buffered saline. Antibodies were then added and incubated overnight (1:1000 each of mouse anti-rat monoclonal CD31 (Ab64543) (Abcam, Cambridge, UK), rabbit anti-rat polyclonal CD68 (Ab125212) (Abcam), mouse anti-rat monoclonal CD163 (MCA342GA) (BioRad, Feldkirchen, Germany), and rabbit anti-human monoclonal Ku80 (EPR3468) (Abcam)). After washing, endogenous peroxidase was blocked in 3% HO. Secondary biotinylated antibodies were then added for 30 min (1:100 anti-mouse and anti-rabbit Ig (RPN1001V, RPN1004V1) (GE-healthcare, Solingen, Germany). 1% streptavidin peroxidase (GE-healthcare) was then added. Histogreen was used as a substrate developer (Linaris GmbH, Dossenheim, Germany). Nuclei were counterstained with Mayer's hematoxylin, and sections were dehydrated and mounted with 99% ethanol, tissue clear, and n-butyl acetate. Control slides were left unstained to assess Histogreen background signal or stained with secondary antibody alone. Slides were scanned (Zeiss AXIO Scan.Z1) and analyzed using QuPath open software (36), which created color filters to quantify the Histogreen-positive area throughout the wound, predefined as a region of interest.

[0095] hKu80 staining of the organs was verified using Alexa Fluor 488-conjugated secondary antibody or Alexa Fluor 568-conjugated secondary antibody (1:1000; Life Technologies, Thermo Fisher Scientific) and TO-PRO-3 nuclear staining (Thermo Fisher Scientific), and evaluated by confocal microscopy.

[0096] Histological scoring system Epidermal thickness index (ETI) At 14-day wounds, the mean thickness of the wound epidermis was calculated at five positions and compared to the mean thickness of the non-lesional epidermis. ETI = mean epidermal thickness of the wound area × 100 / mean epidermal thickness of non-injured skin

[0097] ETI > 105% is considered hypertrophic and is mostly observed during the re-epithelialization phase, indicating healing. The return of the epidermal thickness close to that of non-injured skin (95% < ETI < 105%) is only observed after the remodeling phase (39).

[0098] Scar elevation index (SEI) At 14-day wounds, the mean thickness of the dermis was calculated using five regions and compared to the mean thickness of the non-wounded dermis. SEI = mean dermal thickness of the wound area × 100 / mean dermal thickness of non-injured skin

[0099] The hypertrophic dermis of the wound (SEI > 105%) can reflect excessive collagen deposition and thus serves as an indirect indicator of scar formation. The atrophic dermis with SEI < 95% is generally reported in the early stages of wound healing and reflects underdevelopment of the dermis. 95% < SEI < 105% characterizes the wound dermis with normal thickness and is only observed at the final stage of healing (39).

[0100] Chip-based dPCR for detecting residual human cells To track the fate of locally applied hMSCs, human DNA in wounds and organs was analyzed using a highly sensitive dPCR method (40).

[0101] dPCR assays were designed for the detection of the single-locus gene GAPDH in the rat and human genomes using specific primers and TaqMan™ probes with minor groove binding (MGB) modifications at the 3' end. For human GAPDH: forward primer, 5'-ccccacacacatgcacttacc-3'; reverse primer, 5'-cctagtcccagggctttgatt-3'; VIC-labeled probe, 5'-taggaaggacaggcaac-3'; for mouse / rat GAPDH: forward primer, 5'-gaatataaaattagatctctttggac-3'; reverse primer, 5'-gttgaatgcttggatgtacaacc-3'; FAM-labeled probe, 5'-taggaaggacaggcaac-3'. Human / rat GAPDH assays were prepared as 40-fold concentrated mixtures containing 9 μmol of each primer and 5 μmol of each probe, giving final concentrations of 225 nmol of each primer and 125 nmol of each probe.

[0102] dPCR (QuantStudio™ 3D; Thermo Fisher Scientific) was performed on a chip with 20,000 reaction wells, each with a volume of 755 pL. For each dPCR analysis, 7.1 μL of DNA was mixed with 0.375 μL of 40× GAPDH assay and 7.5 μL of dPCR Master Mix V2 (Thermo Fisher Scientific) containing ROX as the reference dye. The cycling program started with 96°C for 10 minutes, followed by 40 cycles of 98°C for 30 seconds and 52°C for 2 minutes. After cycling, the dPCR chip was scanned for FAM and VIC signals (QuantStudio™ 3D Chip Reader; Thermo Fisher Scientific), and the data were analyzed using QuantStudio 3D AnalysisSuite cloud software (https: / / apps.thermofisher.com / quantstudio3d). Based on the fluorescent signal and statistical correction using a Poisson distribution, the software was able to calculate the target copy number per μL and the target / total (%) value. To validate the assay, pure human and rat genomic DNA were mixed at defined ratios (1:10, 1:20, and 1:50). Human DNA was reliably detectable in the 1:50 mixture (detection limit 2%; approximately 4 copies / μL), while pure rat DNA showed a background signal of 0.2% (approximately 0.4 copies / μL). 0.5 copies / μL was calculated as the cutoff for a positive signal.

[0103] Quantitative data are presented as mean ± standard deviation (SD) and compared by analysis of variance (ANOVA) and post-hoc tests as indicated using GraphPad Prism (La Jolla, CA, USA). A value of p < 0.05 was considered statistically significant.

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Claims

1. 1. A method for generating a donor cell pool of expandable cells derived from bone marrow samples of multiple genetically non-identical bone marrow donors, comprising: (a) providing two or more bone marrow donor samples from genetically non-identical (or different) bone marrow donors; (b) separately passaging bone marrow-derived cells from each bone marrow donor sample of (a) into (separate) cell culture receptacles such as culture flasks or dishes, or bioreactors (p0); (c) culturing bone marrow-derived cells from each donor separately; (d) a step (p1) of separately subculturing a portion of each culture of bone marrow-derived cells from (c) into a new cell culture receptacle such as a culture flask or dish; (e) culturing bone marrow-derived cells from each donor separately; (f) Passaging and pooling a portion of each culture of bone marrow-derived cells from (e) into a single cell culture receptacle to obtain a donor cell pool (p2); A method comprising:

2. The method of claim 1, further comprising, following (f), a step (f') of cryopreserving the donor cell pool as a working cell bank.

3. 3. The method of claim 1 or 2, further comprising a step (g) of culturing the donor cell pool either directly from step (f) or after thawing a cell sample of the working cell bank of claim 2.

4. The method according to any one of claims 1 to 3, further comprising a step (h) (p3) of passaging a portion of the cells cultured and pooled in step (g) into (separate) cell culture receptacles.

5. 5. The method of claim 4, wherein the cultured cells of claim 4 are obtained as a clinical product suitable for medical use.

6. The method according to any one of claims 1 to 5, wherein passaging comprises transferring a portion of the cultured cells from the cell culture receptacle to a new receptacle containing fresh cell culture medium.

7. The method of any one of claims 1 to 6, wherein the donor is a human donor.

8. 8. The method according to any one of claims 1 to 7, wherein between p0 and p1, a portion of the cells are transferred to a storage receptacle and frozen to obtain a donor cell master cell bank, and for continuing p1, the cells in the storage receptacle are thawed and passaged into a cell culture receptacle, and further cell cultured in step (c).

9. A working cell bank and / or clinical product produced or obtainable by the method of any one of claims 1 to 12.

10. A preparation of clinical grade pooled mesenchymal stromal cells (MSCs) obtainable by the method of any one of claims 1 to 12.

11. 13. A preparation of clinical-grade pooled mesenchymal stromal cells (MSCs) for use in treating a disease or condition in a subject, the preparation being obtained or obtainable by a method according to any one of claims 1 to 12.

12. 12. A method for producing a therapeutically active composition, the method comprising carrying out a method according to any one of claims 1 to 8, or providing a working cell bank and / or clinical product according to claim 9, or a preparation according to claim 10 or 11, culturing and / or expanding MSC material in cell culture, and harvesting a composition in which the MSC material has secreted one or more cellular factors, thereby obtaining the therapeutically active composition.

13. 13. The method of claim 12, wherein the therapeutically active composition comprises one or more growth factors and / or other bioreactive molecules, preferably selected from brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), granulocyte-colony stimulating factor (G-CSF), hepatocyte growth factor (HGF), IL-1a, IL-6, LIF, osteopontin, vascular endothelial growth factor (VEGF)-A, fibroblast growth factor (FGF-2), transforming growth factor (TGF)-b, prostaglandin E (PG) E-2, and indoleamine 2,3-dioxygenase (IDO) after priming; and / or wherein the therapeutically active composition comprises MSC-derived extracellular vesicles (ECVs).

14. A therapeutically active composition, such as a cell-free composition, obtained or obtainable by the method of claim 12 or 13.

15. 15. The therapeutically active composition of claim 14, which is a pharmaceutical composition further comprising one or more pharmaceutically acceptable carriers and / or excipients.

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