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

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

Scientific Reports ◽

10.1038/s41598-021-83088-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Samatha Bhat ◽

Pachaiyappan Viswanathan ◽

Shashank Chandanala ◽

S. Jyothi Prasanna ◽

Raviraja N. Seetharam

Keyword(s):

Bone Marrow ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Population Doubling ◽

Seeding Density ◽

Population Doubling Time ◽

Differentiation Potential ◽

Serum Free ◽

Safety Issues ◽

Free Media

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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The Osteogenic Properties of Multipotent Mesenchymal Stromal Cells in Cultures on TiO2Sol-Gel-Derived Biomaterial

BioMed Research International ◽

10.1155/2015/651097 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 10

Author(s):

Krzysztof Marycz ◽

Agnieszka Śmieszek ◽

Jakub Grzesiak ◽

Anna Siudzińska ◽

Monika Marędziak ◽

...

Keyword(s):

Osteogenic Differentiation ◽

Stromal Cells ◽

Sol Gel ◽

Multipotent Mesenchymal Stromal Cells ◽

Biological Evaluation ◽

Population Doubling ◽

Population Doubling Time ◽

Multipotent Stromal Cells ◽

Differentiation Potential ◽

Characteristic Features

The biocompatibility of the bone implants is a crucial factor determining the successful tissue regeneration. The aim of this work was to compare cellular behavior and osteogenic properties of rat adipose-derived multipotent stromal cells (ASCs) and bone marrow multipotent stromal cells (BMSCs) cultured on metallic substrate covered with TiO2sol-gel-derived nanolayer. The morphology, proliferation rate, and osteogenic differentiation potential of both ASCs and BMSCs propagated on the biomaterials were examined. The potential for osteogenic differentiation of ASCs and BMSCs was determined based on the presence of specific markers of osteogenesis, that is, alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCL). Additionally, the concentration of calcium and phosphorus in extracellular matrix was determined using energy-dispersive X-ray spectroscopy (SEM-EDX). Obtained results showed that TiO2layer influenced proliferation activity of ASCs, which manifested by shortening of population doubling time and increase of OPN secretion. However, characteristic features of cells morphology and growth pattern of cultures prompted us to conclude that ultrathin TiO2layer might also enhance osteodifferentiation of BMSCs. Therefore in our opinion, both populations of MSCs should be used for biological evaluation of biomaterials compatibility, such results may enhance the area of investigations related to regenerative medicine.

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Human Acquired Aplastic Anemia Patients’ Bone-Marrow-Derived Mesenchymal Stem Cells Are Not Influenced by Hematopoietic Compartment and Maintain Stemness and Immune Properties

Anemia ◽

10.1155/2021/6678067 ◽

2021 ◽

Vol 2021 ◽

pp. 1-9

Author(s):

Vandana Sharma ◽

Sonali Rawat ◽

Suchi Gupta ◽

Sweta Tamta ◽

Rinkey Sharma ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Aplastic Anemia ◽

Bone Marrow Failure ◽

Population Doubling ◽

Population Doubling Time ◽

Differentiation Potential ◽

Proliferation Capacity ◽

Trilineage Differentiation

Background and Objective. Acquired aplastic anemia (aAA) is a bone marrow failure disorder characterized by pancytopenia and bone marrow aplasia. Bone marrow Mesenchymal Stem Cells (BM-MSCs) are an important component of BM microenvironment, associated with hematopoietic and immune homeostasis. Any alterations in BM microenvironment can disrupt the normal functioning and it needs to be assessed. Methods. In the current study, we investigated the morphological differences, proliferation capacity, population doubling time (PDT), surface marker profiling, trilineage differentiation potential, and immunosuppressive ability of BM Mesenchymal Stem Cells (BM-MSCs) from untreated aAA patients and in the same number of age- and gender-matched controls. Results. We observed similar morphology, proliferation capacity, phenotype, trilineage differentiation potential, and immunomodulatory properties of BM-MSCs in aAA patients and control subjects. Conclusion. Our results confirm that the basic and immunosuppressive properties of BM-MSCs from aAA patients do not differ from normal BM-MSCs. Our data suggest that BM-MSCs from aAA patients might not be involved in disease pathogenesis. However, owing to a smaller number of samples, it is not conclusive, and future studies with more exhaustive investigation at transcriptome level are warranted.

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POSTNATAL EXTRA-EMBRYONIC TISSUES AS A SOURCE OF MULTIPLE CELL TYPES FOR REGENERATIVE MEDICINE APPLICATIONS

Experimental Oncology ◽

10.31768/2312-8852.2017.39(3):186-190 ◽

2017 ◽

Vol 39 (3) ◽

pp. 186-190 ◽

Cited By ~ 1

Author(s):

O S Gubar ◽

A I Rodnichenko ◽

R G Vasylie ◽

A V Zlatska ◽

D O Zubov

Keyword(s):

Regenerative Medicine ◽

Umbilical Cord ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Umbilical Vein ◽

Cell Types ◽

Population Doubling ◽

Embryonic Tissues ◽

Chorionic Membrane ◽

Multiple Cell

Aim: We aimed to isolate and characterize the cell types which could be obtained from postnatal extra-embryonic tissues. Materials and Methods: Fresh tissues (no more than 12 h after delivery) were used for enzymatic or explants methods of cell isolation. Obtained cultures were further maintained at 5% oxygen. At P3 cell phenotype was assessed by fluorescence-activated cell sorting, population doubling time was calculated and the multilineage differentiation assay was performed. Results: We have isolated multiple cell types from postnatal tissues. Namely, placental mesenchymal stromal cells from placenta chorionic disc, chorionic membrane mesenchymal stromal cells (ChM-MSC) from free chorionic membrane, umbilical cord MSC (UC-MSC) from whole umbilical cord, human umbilical vein endothelial cells (HUVEC) from umbilical vein, amniotic epithelial cells (AEC) and amniotic MSC (AMSC) from amniotic membrane. All isolated cell types displayed high proliferation rate together with the typical MSC phenotype: CD73+CD90+CD105+CD146+CD166+CD34-CD45-HLA-DR-. HUVEC constitutively expressed key markers CD31 and CD309. Most MSC and AEC were capable of osteogenic and adipogenic differentiation. Conclusion: We have shown that a wide variety of cell types can be easily isolated from extra-embryonic tissues and expanded ex vivo for regenerative medicine applications. These cells possess typical MSC properties and can be considered an alternative for adult MSC obtained from bone marrow or fat, especially for allogeneic use.

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Establishment and characterization of fibroblast cultures derived from a female common hippopotamus (Hippopotamus amphibius) skin biopsy

10.1101/2020.10.14.338632 ◽

2020 ◽

Author(s):

Tao Wang ◽

Zelong Li ◽

Jinpu Wei ◽

Dongmin Zheng ◽

Chen Wang ◽

...

Keyword(s):

Cell Cultures ◽

Cultured Cells ◽

Population Decline ◽

Fibroblast Cell ◽

Population Doubling ◽

Population Doubling Time ◽

Fibroblast Cultures ◽

Hippopotamus Amphibius ◽

The Common

AbstractThe population decline in the common hippopotamus (Hippopotamus amphibius) has necessitated the preservation of their genetic resources for species conservation and research. Of all actions, cryopreservation of fibroblast cell cultures derived from animal biopsy is considered a simple but efficient means. Nevertheless, preserving viable cell cultures of the common hippopotamus has not been achieved to our knowledge. To this end, we detailed a method to establish fibroblast cell cultures from a female common hippopotamus fetus in this study. By combining the classic tissue explant direct culture and enzymatic digestion methods, we isolated a great number of cells with typical fibroblastic morphology and high viability. Characterization of the fibroblast cultures was carried out using different techniques. In short, neither bacteria/fungi nor mycoplasma was detectable in the cell cultures throughout the study. The population doubling time was 23.9 h according to the growth curve. Karyotyping based on Giemsa staining showed that cultured cells were diploid with 36 chromosomes in all, one pair of which was sex chromosomes. Mitochondrial cytochrome C oxidase subunit I gene sequence of the cultured cells was 99.26% identical with the Hippopotamus amphibius complete mitochondrial DNA sequence registered in GenBank, confirming the cells were derived from a common hippopotamus. Flow cytometry and immunofluorescence staining results revealed that the detected cells were positive for fibroblast markers, S100A4 and Vimentin. In conclusion, we isolated and characterized a new fibroblast cell culture from a common hippopotamus skin sample and the cryopreserved cells could be useful genetic materials for the future research.

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The Treasury of Wharton's Jelly

Stem Cell Reviews and Reports ◽

10.1007/s12015-021-10217-8 ◽

2021 ◽

Author(s):

Rebecca Guenther ◽

Stephan Dreschers ◽

Jessika Maassen ◽

Daniel Reibert ◽

Claudia Skazik-Voogt ◽

...

Keyword(s):

Stem Cells ◽

Umbilical Cord ◽

Cultured Cells ◽

Single Cells ◽

Mesenchymal Progenitor Cells ◽

Differentiation Potential ◽

Lineage Differentiation ◽

Umbilical Cord Tissue ◽

The Common ◽

Culture Supernatants

Abstract Background Postnatal umbilical cord tissue contains valuable mesenchymal progenitor cells of various differentiation stages. While mesenchymal stem cells are plastic-adherent and tend to differentiate into myofibroblastic phenotypes, some round cells detach, float above the adherent cells, and build up cell aggregates, or form spheroids spontaneously. Very small luminescent cells are always involved as single cells or within collective forms and resemble the common well-known very small embryonic-like cells (VSELs). In this study, we investigated these VSELs-like cells in terms of their pluripotency phenotype and tri-lineage differentiation potential. Methods VSELs-like cells were isolated from cell-culture supernatants by a process that combines filtering, up concentration, and centrifugation. To determine their pluripotency character, we measured the expression of Nanog, Sox-2, Oct-4, SSEA-1, CXCR4, SSEA-4 on gene and protein level. In addition, the cultured cells derived from UC tissue were examined regarding their potential to differentiate into three germ layers. Result The VSELs-like cells express all of the pluripotency-associated markers we investigated and are able to differentiate into meso- endo- and ectodermal precursor cells. Conclusions Umbilical cord tissue hosts highly potent VSELs-like stem cells. Graphical Abstract

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Low Oxygen Tension Potentiates Proliferation and Stemness but Not Multilineage Differentiation of Caprine Male Germline Stem Cells

10.21203/rs.3.rs-420611/v1 ◽

2021 ◽

Author(s):

Shiva Pratap Singh ◽

Suresh Dinkar Kharche ◽

Manisha Pathak ◽

Ravi Ranjan ◽

Yogesh Kumar Soni ◽

...

Keyword(s):

Stem Cells ◽

Ex Vivo ◽

Growth Characteristics ◽

Population Doubling ◽

Population Doubling Time ◽

Germline Stem Cells ◽

Multilineage Differentiation ◽

Low Oxygen ◽

Differentiation Potential

Abstract The milieu of testicular germline stem cells (mGSCs) is characterized as low oxygen (O2) environment, whereas, there in-vitro expansion is typically performed under normoxia (20-21% O2). Here, we evaluated and compared the culture and multilineage differentiation characteristics of enriched (through differential platting and percoll density centrifugation) caprine mGSCs (cmGSCs) under hypoxic (5% O2) and normoxic (21% O2) culture conditions. For this, in addition to growth characteristics and population-doubling time (PDT); viability, proliferation, senescence, and expression of key-markers of adhesion (β-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated and compared under normoxia and hypoxia. Moreover, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) was assessed. The survival, viability and proliferation were significantly promoted and PDT was reduced (p < 0.05), thus yielding a higher number of viable cells with larger colonies under hypoxia. Furthermore, expression of stemness and adhesion markers was distinctly increased under lowered O2 condition. Conversely, the presence of differentiated regions and expression of differentiation specific key genes [C/EBPα (adipogenic), nestin and β-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p < 0.05) reduced under hypoxic conditions. These data demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics, and stemness but not the multilineage differentiation potential of cmGSCs as compared with normoxia. These data are important for the development of robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.

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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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Perspectives for Future Use of Extracellular Vesicles from Umbilical Cord- and Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells in Regenerative Therapies—Synthetic Review

International Journal of Molecular Sciences ◽

10.3390/ijms21030799 ◽

2020 ◽

Vol 21 (3) ◽

pp. 799 ◽

Cited By ~ 7

Author(s):

Joanna Lelek ◽

Ewa K. Zuba-Surma

Keyword(s):

Adipose Tissue ◽

Umbilical Cord ◽

Extracellular Vesicles ◽

Stromal Cells ◽

Tissue Repair ◽

Molecular Mechanisms ◽

Skin Diseases ◽

Differentiation Potential

Mesenchymal stem/ stromal cells (MSCs) represent progenitor cells of various origin with multiple differentiation potential, representing the most studied population of stem cells in both in vivo pre-clinical and clinical studies. MSCs may be found in many tissue sources including extensively studied adipose tissue (ADSCs) and umbilical cord Wharton’s jelly (UC-MSCs). Most of sanative effects of MSCs are due to their paracrine activity, which includes also release of extracellular vesicles (EVs). EVs are small, round cellular derivatives carrying lipids, proteins, and nucleic acids including various classes of RNAs. Due to several advantages of EVs when compare to their parental cells, MSC-derived EVs are currently drawing attention of several laboratories as potential new tools in tissue repair. This review focuses on pro-regenerative properties of EVs derived from ADSCs and UC-MSCs. We provide a synthetic summary of research conducted in vitro and in vivo by employing animal models and within initial clinical trials focusing on neurological, cardiovascular, liver, kidney, and skin diseases. The summarized studies provide encouraging evidence about MSC-EVs pro-regenerative capacity in various models of diseases, mediated by several mechanisms. Although, direct molecular mechanisms of MSC-EV action are still under investigation, the current growing data strongly indicates their potential future usefulness for tissue repair.

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