Expansion and characterization of bone marrow derived human mesenchymal stromal cells in serum-free conditions

AbstractBone marrow-derived mesenchymal stromal cells (BM-MSCs) are gaining increasing importance in the field of regenerative medicine. Although therapeutic value of MSCs is now being established through many clinical trials, issues have been raised regarding their expansion as per regulatory guidelines. Fetal bovine serum usage in cell therapy poses difficulties due to its less-defined, highly variable composition and safety issues. Hence, there is a need for transition from serum-based to serum-free media (SFM). Since SFM are cell type-specific, a precise analysis of the properties of MSCs cultured in SFM is required to determine the most suitable one. Six different commercially available low serum/SFM with two different seeding densities were evaluated to explore their ability to support the growth and expansion of BM-MSCs and assess the characteristics of BM-MSCs cultured in these media. Except for one of the SFM, all other media tested supported the growth of BM-MSCs at a low seeding density. No significant differences were observed in the expression of MSC specific markers among the various media tested. In contrary, the population doubling time, cell yield, potency, colony-forming ability, differentiation potential, and immunosuppressive properties of MSCs varied with one another. We show that SFM tested supports the growth and expansion of BM-MSCs even at low seeding density and may serve as possible replacement for animal-derived serum.

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Human bone marrow-derived mesenchymal stromal cells cultured in serum-free media demonstrate enhanced antifibrotic abilities via prolonged survival and robust regulatory T cell induction in murine bleomycin-induced pulmonary fibrosis

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02574-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Shun Takao ◽

Taku Nakashima ◽

Takeshi Masuda ◽

Masashi Namba ◽

Shinjiro Sakamoto ◽

...

Keyword(s):

Bone Marrow ◽

Pulmonary Fibrosis ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Pulmonary Inflammation ◽

Human Bone ◽

Human Bone Marrow ◽

Distributed Data ◽

Serum Free ◽

Free Media

Abstract Background Mesenchymal stromal cells (MSCs) are a potential therapeutic tool for pulmonary fibrosis. However, ex vivo MSC expansion using serum poses risks of harmful immune responses or unknown pathogen infections in the recipients. Therefore, MSCs cultured in serum-free media (SF-MSCs) are ideal for clinical settings; however, their efficacy in pulmonary fibrosis is unknown. Here, we investigated the effects of SF-MSCs on bleomycin-induced pulmonary inflammation and fibrosis compared to those of MSCs cultured in serum-containing media (S-MSCs). Methods SF-MSCs and S-MSCs were characterized in vitro using RNA sequence analysis. The in vivo kinetics and efficacy of SF-MSC therapy were investigated using a murine model of bleomycin-induced pulmonary fibrosis. For normally distributed data, Student’s t test and one-way repeated measures analysis of variance followed by post hoc Tukey’s test were used for comparison between two groups and multiple groups, respectively. For non-normally distributed data, Kruskal–Wallis and Mann–Whitney U tests were used for comparison between groups, using e Bonferroni’s correction for multiple comparisons. All tests were two-sided, and P < 0.05 was considered statistically significant. Results Serum-free media promoted human bone marrow-derived MSC expansion and improved lung engraftment of intravenously administered MSCs in recipient mice. SF-MSCs inhibited the reduction in serum transforming growth factor-β1 and the increase of interleukin-6 in both the serum and the bronchoalveolar lavage fluid during bleomycin-induced pulmonary fibrosis. SF-MSC administration increased the numbers of regulatory T cells (Tregs) in the blood and lungs more strongly than in S-MSC administration. Furthermore, SF-MSCs demonstrated enhanced antifibrotic effects on bleomycin-induced pulmonary fibrosis, which were diminished by antibody-mediated Treg depletion. Conclusions SF-MSCs significantly suppressed BLM-induced pulmonary inflammation and fibrosis through enhanced induction of Tregs into the lungs and corrected the dysregulated cytokine balance. Therefore, SF-MSCs could be a useful tool for preventing pulmonary fibrosis progression without the demerits of serum use.

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Comparative Analysis of Biological and Functional Properties of Bone Marrow Mesenchymal Stromal Cells Expanded in Media with Different Platelet Lysate Content

Cells Tissues Organs ◽

10.1159/000492581 ◽

2018 ◽

Vol 205 (4) ◽

pp. 226-239 ◽

Cited By ~ 1

Author(s):

Marijana Skific ◽

Mirna Golemovic ◽

Kristina Crkvenac-Gornik ◽

Radovan Vrhovac ◽

Branka Golubic Cepulic

Keyword(s):

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Immunological Tolerance ◽

Biological Properties ◽

Platelet Lysate ◽

Population Doubling ◽

Population Doubling Time ◽

Significant Difference

Due to their ability to induce immunological tolerance in the recipient, mesenchymal stromal cells (MSCs) have been utilized in the treatment of various hematological and immune- and inflammation-mediated diseases. The clinical application of MSCs implies prior in vitro expansion that usually includes the use of fetal bovine serum (FBS). The present study evaluated the effect of different platelet lysate (PL) media content on the biological properties of MSCs. MSCs were isolated from the bone marrow of 13 healthy individuals and subsequently expanded in three different culture conditions (10% PL, 5% PL, 10% FBS) during 4 passages. The cells cultured in different conditions had comparable immunophenotype, clonogenic potential, and differentiation capacity. However, MSC growth was significantly enhanced in the presence of PL. Cultures supplemented with 10% PL had a higher number of cumulative population doublings in all passages when compared to the 5% PL condition (p < 0.03). Such a difference was also observed when 10% PL and 10% FBS conditions were compared (p < 0.005). A statistically significant difference in population doubling time was determined only between the 10% PL and 10% FBS conditions (p < 0.005). Furthermore, MSCs cultured in 10% PL were able to cause a 66.9% reduction of mitogen-induced lymphocyte proliferation. Three chromosome aberrations were detected in PL conditions. Since two changes occurred in the same do nor, it is possible they were donor dependent rather than caused by the culture condition. These findings demonstrate that a 10% PL condition enables a higher yield of MSCs within a shorter time without altering MSC properties, and should be favored over the 5% PL condition.

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Comparative Bioactivity Analysis for Off-the-Shelf and Culture–Rescued Umbilical Cord-Derived Mesenchymal Stem/Stromal Cells in a Xeno- and Serum-Free Culture System

Cell Transplantation ◽

10.1177/09636897211039441 ◽

2021 ◽

Vol 30 ◽

pp. 096368972110394

Author(s):

Minh Quang Nguyen ◽

Hue T. H. Bui ◽

Anh Nguyen Thi Tuyet ◽

Trinh Thi Hong Nhung ◽

Duc M. Hoang ◽

...

Keyword(s):

Umbilical Cord ◽

Stromal Cells ◽

Cultured Cells ◽

Cost Effective ◽

Population Doubling ◽

Population Doubling Time ◽

Differentiation Potential ◽

Serum Free ◽

Proliferation Capacity ◽

Cell Growth And Proliferation

We recently reported a standardized xeno- and serum-free culture platform to isolate and expand umbilical cord-derived mesenchymal stem/stromal cells (UC-MSCs). Comparing populations from the same passage, cells that were cryopreserved and culture-rescued exhibited characteristics similar to those of their fresh counterparts, continuously cultured cells without interim cryopreservation. The culture rescue after thawing allowed for the cells to be fully recovered. However, since it would be more cost-effective and timesaving if cryopreserved cells can be used as an off-the-shelf product, we set out to compare the bioactivity of freshly thawed UC-MSCs versus culture-rescued UC-MSCs of the same batch that were recultured for an additional passage under our xeno- and serum-free protocol. UC-MSCs showed high viability in both the freshly thawed and the re-cultured group. Both populations displayed a similar proliferation capacity which is indicated by a comparable population doubling time and colony-forming ability. Both freshly thawed and culture-rescued UC-MSCs expressed the characteristic immunophenotype and were capable of differentiating into osteocytes, chondrocytes, and adipocytes. On the other hand, culture-rescued cells appeared to be more potent in immunosuppression than freshly thawed cells. In conclusion, freshly thawed and culture-rescued cell products share comparable bioactivity in cell growth and proliferation, immunophenotype, and differentiation potential. However, the culture-rescued cells that were allowed to grow for an additional passage appear to display a more favorable immunomodulatory potential when compared to their freshly thawed parent cells.

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Evaluation of Tissue Homogenization to Support the Generation of GMP-Compliant Mesenchymal Stromal Cells from the Umbilical Cord

Stem Cells International ◽

10.1155/2016/3274054 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 6

Author(s):

Ryan J. Emnett ◽

Aparna Kaul ◽

Aleksandar Babic ◽

Vicki Geiler ◽

Donna Regan ◽

...

Keyword(s):

Umbilical Cord ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Cultured Cells ◽

Good Manufacturing Practice ◽

Population Doubling ◽

Population Doubling Time ◽

Differentiation Potential ◽

Clonogenic Potential ◽

Therapeutic Doses

Recent studies have demonstrated that the umbilical cord (UC) is an excellent source of mesenchymal stromal cells (MSCs). However, current protocols for extracting and culturing UC-MSCs do not meet current good manufacturing practice (cGMP) standards, in part due to the use of xenogeneic reagents. To support the development of a cGMP-compliant method, we have examined an enzyme-free isolation method utilizing tissue homogenization (t-H) followed by culture in human platelet lysate (PL) supplemented media. The yield and viability of cells after t-H were comparable to those obtained after collagenase digestion (Col-D). Importantly, kinetic analysis of cultured cells showed logarithmic growth over 10 tested passages, although the rate of cell division was lower for t-H as compared to Col-D. This slower growth of t-H-derived cells was also reflected in their longer population doubling time. Interestingly, there was no difference in the expression of mesenchymal markers and trilineage differentiation potential of cells generated using either method. Finally, t-H-derived cells had greater clonogenic potential compared to Col-D/FBS but not Col-D/PL and were able to maintain CFU-F capacity through P7. This bench scale study demonstrates the possibility of generating therapeutic doses of good quality UC-MSCs within a reasonable length of time using t-H and PL.

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Successful scale up expansion of Wharton's jelly mesenchymal stromal cells in different commercial xeno-free and serum-free media

Cytotherapy ◽

10.1016/j.jcyt.2020.03.162 ◽

2020 ◽

Vol 22 (5) ◽

pp. S94

Author(s):

A. López Fernández ◽

I. Carreras Sánchez ◽

J. Vives

Keyword(s):

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Scale Up ◽

Wharton’S Jelly ◽

Serum Free ◽

Wharton's Jelly ◽

Free Media

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Heparin Anticoagulant for Human Bone Marrow Does Not Influence In Vitro Performance of Human Mesenchymal Stromal Cells

Cells ◽

10.3390/cells9071580 ◽

2020 ◽

Vol 9 (7) ◽

pp. 1580

Author(s):

Yvonne Roger ◽

Laura Burmeister ◽

Anika Hamm ◽

Kirsten Elger ◽

Oliver Dittrich-Breiholz ◽

...

Keyword(s):

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Mononuclear Cells ◽

Population Doubling ◽

Pcr Analysis ◽

Inhibitory Influence ◽

In Vitro Differentiation ◽

Cell Surface Antigens

Mesenchymal stromal cells (MSCs) are a promising cell source for tissue engineering and regenerative medicine. In our lab, we found that MSC preparations from bone marrow of many different donors had a limited capacity of in vitro differentiation into osteogenic and chondrogenic lineages—a capacity claimed to be inherent to MSCs. The current study was designed to test the hypothesis that the amount of heparin used as anticoagulant during bone marrow harvest had an inhibitory influence on the in vitro differentiation capacity of isolated MSCs. Bone marrow was obtained from the femoral cavity of twelve donors during total hip arthroplasty in the absence or presence of heparin. No coagulation was observed in the absence of heparin. The number of mononuclear cells was independent of heparin addition. Isolated MSCs were characterized by morphology, population doubling times, expression of cell surface antigens and in vitro differentiation. Results of these analyses were independent of the amount of heparin. Transcriptome analyses of cells from three randomly chosen donors and quantitative realtime PCR (qRT-PCR) analysis from cells of all donors demonstrated no clear effect of heparin on the transcriptome of the cells. This excludes heparin as a potential source of disparate results.

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Culture and characterization of various porcine integumentary-connective tissue-derived mesenchymal stromal cells to facilitate tissue adhesion to percutaneous metal implants

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02666-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Devaveena Dey ◽

Nicholas G. Fischer ◽

Andrea H. Dragon ◽

Elsa Ronzier ◽

Isha Mutreja ◽

...

Keyword(s):

Bone Marrow ◽

Connective Tissue ◽

Achilles Tendon ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Soft Tissues ◽

Flexor Tendon ◽

Cytoskeletal Proteins ◽

Preclinical Research ◽

Differentiation Potential

Abstract Background Transdermal osseointegrated prosthesis have relatively high infection rates leading to implant revision or failure. A principle cause for this complication is the absence of a durable impervious biomechanical seal at the interface of the hard structure (implant) and adjacent soft tissues. This study explores the possibility of recapitulating an analogous cellular musculoskeletal-connective tissue interface, which is present at naturally occurring integumentary tissues where a hard structure exits the skin, such as the nail bed, hoof, and tooth. Methods Porcine mesenchymal stromal cells (pMSCs) were derived from nine different porcine integumentary and connective tissues: hoof-associated superficial flexor tendon, molar-associated periodontal ligament, Achilles tendon, adipose tissue and skin dermis from the hind limb and abdominal regions, bone marrow and muscle. For all nine pMSCs, the phenotype, multi-lineage differentiation potential and their adhesiveness to clinical grade titanium was characterized. Transcriptomic analysis of 11 common genes encoding cytoskeletal proteins VIM (Vimentin), cell–cell and cell–matrix adhesion genes (Vinculin, Integrin β1, Integrin β2, CD9, CD151), and for ECM genes (Collagen-1a1, Collagen-4a1, Fibronectin, Laminin-α5, Contactin-3) in early passaged cells was performed using qRT-PCR. Results All tissue-derived pMSCs were characterized as mesenchymal origin by adherence to plastic, expression of cell surface markers including CD29, CD44, CD90, and CD105, and lack of hematopoietic (CD11b) and endothelial (CD31) markers. All pMSCs differentiated into osteoblasts, adipocytes and chondrocytes, albeit at varying degrees, under specific culture conditions. Among the eleven adhesion genes evaluated, the cytoskeletal intermediate filament vimentin was found highly expressed in pMSC isolated from all tissues, followed by genes for the extracellular matrix proteins Fibronectin and Collagen-1a1. Expression of Vimentin was the highest in Achilles tendon, while Fibronectin and Col1agen-1a1 were highest in molar and hoof-associated superficial flexor tendon bone marrow, respectively. Achilles tendon ranked the highest in both multilineage differentiation and adhesion assessments to titanium metal. Conclusions These findings support further preclinical research of these tissue specific-derived MSCs in vivo in a transdermal osseointegration implant model.

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Augmenting emergency granulopoiesis with CpG conditioned mesenchymal stromal cells in murine neutropenic sepsis

Blood Advances ◽

10.1182/bloodadvances.2020002556 ◽

2020 ◽

Vol 4 (19) ◽

pp. 4965-4979 ◽

Cited By ~ 1

Author(s):

Julie Ng ◽

Fei Guo ◽

Anna E. Marneth ◽

Sailaja Ghanta ◽

Min-Young Kwon ◽

...

Keyword(s):

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Therapeutic Potential ◽

Neutrophil Extracellular Trap ◽

Differentiation Potential ◽

Neutropenic Sepsis ◽

Cpg Oligodeoxynucleotide ◽

Increased Risk

Abstract Patients with immune deficiencies from cancers and associated treatments represent a growing population within the intensive care unit with increased risk of morbidity and mortality from sepsis. Mesenchymal stromal cells (MSCs) are an integral part of the hematopoietic niche and express toll-like receptors, making them candidate cells to sense and translate pathogenic signals into an innate immune response. In this study, we demonstrate that MSCs administered therapeutically in a murine model of radiation-associated neutropenia have dual actions to confer a survival benefit in Pseudomonas aeruginosa pneumo-sepsis that is not from improved bacterial clearance. First, MSCs augment the neutrophil response to infection, an effect that is enhanced when MSCs are preconditioned with CpG oligodeoxynucleotide, a toll-like receptor 9 agonist. Using cytometry by time of flight, we identified proliferating neutrophils (Ly6GlowKi-67+) as the main expanded cell population within the bone marrow. Further analysis revealed that CpG-MSCs expand a lineage restricted progenitor population (Lin−Sca1+C-kit+CD150−CD48+) in the bone marrow, which corresponded to a doubling in the myeloid proliferation and differentiation potential in response to infection compared with control. Despite increased neutrophils, no reduction in organ bacterial count was observed between experimental groups. However, the second effect exerted by CpG-MSCs is to attenuate organ damage, particularly in the lungs. Neutrophils obtained from irradiated mice and cocultured with CpG-MSCs had decreased neutrophil extracellular trap formation, which was associated with decreased citrullinated H3 staining in the lungs of mice given CpG-MSCs in vivo. Thus, this preclinical study provides evidence for the therapeutic potential of MSCs in neutropenic sepsis.

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