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Effects of enzyme and cryoprotectant concentrations on yield of equine adipose-derived multipotent stromal cells.
Abstract: OBJECTIVE To evaluate effects of various concentrations of collagenase and dimethyl sulfoxide (DMSO) on yield of equine adipose-derived multipotent stromal cells (ASCs) before and after cryopreservation. SAMPLE Supragluteal subcutaneous adipose tissue from 7 Thoroughbreds. PROCEDURES Tissues were incubated with digests containing 0.1%, 0.05%, or 0.025% type I collagenase. Part of each resulting stromal vascular fraction was cryopreserved in 80% fetal bovine serum (FBS), 10% DMSO, and 10% Dulbecco modified Eagle medium F-12 and in 95% FBS and 5% DMSO. Half of each fresh and cryopreserved heterogeneous cell population was not immunophenotyped (unsorted) or was immunophenotyped for CD44, CD105, and major histocompatability complex class II (MHCII; CD44-CD105-MHCII cells and CD44-CD105-MHCII cells). Cell proliferation (cell viability assay), plasticity (CFU frequency), and lineage-specific target gene and oncogene expression (reverse transcriptase PCR assays) were determined in passage 1 cells before and after culture in induction media. RESULTS Digestion with 0.1% collagenase yielded the highest number of nucleated cells. Cell surface marker expression and proliferation rate were not affected by collagenase concentration. Cryopreservation reduced cell expansion rate and CD44-CD105-MHCII CFUs; it also reduced osteogenic plasticity of unsorted cells. However, effects appeared to be unrelated to DMSO concentrations. There were also variable effects on primordial gene expression among cell isolates. CONCLUSIONS AND CLINICAL RELEVANCE Results supported the use of 0.1% collagenase in an adipose tissue digest and 5% DMSO in cryopreservation medium for isolation and cryopreservation, respectively, of equine ASCs. These results may be used as guidelines for standardization of isolation and cryopreservation procedures for equine ASCs.
Publication Date: 2018-09-27 PubMed ID: 30256145DOI: 10.2460/ajvr.79.10.1100Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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This research paper explores how different concentrations of collagenase and dimethyl sulfoxide (DMSO) affect the yield of equine adipose-derived multipotent stromal cells (ASCs) both before and after the process of cryopreservation. The results of the study suggest using 0.1% collagenase and 5% DMSO respectively to optimize the isolation and cryopreservation of equine ASCs.
Objectives and Methodology
- The main objective of the study was to evaluate the effect of varying concentrations of collagenase and dimethyl sulfoxide (DMSO) on the yield of equine ASCs both pre and post cryopreservation.
- For samples, the subcutaneous adipose tissue from seven Thoroughbreds was used.
- The study procedure involved treating tissues with digests containing 0.1%, 0.05%, or 0.025% type I collagenase.
- Part of each resulting stromal vascular fraction was cryopreserved in mixtures of 80% fetal bovine serum (FBS), 10% DMSO, and 10% Dulbecco modified Eagle medium F-12, and in a 95% FBS and 5% DMSO solution.
- Each fresh and cryopreserved cell population was further split into two halves – one not immunophenotyped (unsorted) and the other immunophenotyped for CD44, CD105, and major histocompatibility complex class II (MHCII).
Results
- It was observed that digestion with 0.1% collagenase yielded the highest number of nucleated cells. However, the concentration of collagenase did not affect cell surface marker expression and proliferation rate.
- The process of cryopreservation was found to reduce cell expansion rate and the count of CD44-CD105-MHCII CFUs. It also reduced the osteogenic plasticity of unsorted cells. Nevertheless, these effects were not linked to DMSO concentrations.
- The research also registered variable effects on primordial gene expression among cell isolates.
Conclusions and Clinical Relevance
- The results obtained supported the use of 0.1% collagenase in the digestion of adipose tissue and 5% DMSO in the cryopreservation medium for the isolation and cryopreservation of equine ASCs respectively.
- These findings can be applied as standard guidelines for the procedures of isolation and cryopreservation equine ASCs.
Cite This Article
APA
Duan W, Lopez MJ.
(2018).
Effects of enzyme and cryoprotectant concentrations on yield of equine adipose-derived multipotent stromal cells.
Am J Vet Res, 79(10), 1100-1112.
https://doi.org/10.2460/ajvr.79.10.1100 Publication
Researcher Affiliations
MeSH Terms
- Animals
- Cell Survival / drug effects
- Collagenases / pharmacology
- Cryopreservation / standards
- Cryopreservation / veterinary
- Cryoprotective Agents / pharmacology
- Dimethyl Sulfoxide / pharmacology
- Female
- Horses / anatomy & histology
- Male
- Stromal Cells / cytology
- Stromal Cells / drug effects
- Subcutaneous Fat / cytology
- Subcutaneous Fat / drug effects
Citations
This article has been cited 2 times.- Sharun K, Jambagi K, Kumar R, Gugjoo MB, Pawde AM, Tuli HS, Dhama K, Amarpal. Clinical applications of adipose-derived stromal vascular fraction in veterinary practice. Vet Q 2022 Dec;42(1):151-166.
- Gareev I, Beylerli O, Ilyasova T, Ahmad A, Shi H, Chekhonin V. Therapeutic application of adipose-derived stromal vascular fraction in myocardial infarction. iScience 2024 May 17;27(5):109791.
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