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Abstract BackgroundWhile mesenchymal stromal cell (MSC) therapies show promise for treating several indications due to their regenerative and immunomodulatory capacity, clinical translation has yet to be achieved due to a lack of robust, scalable manufacturing practices. Expansion using undefined fetal bovine serum (FBS) or human platelet lysate contributes to MSC functional heterogeneity and limits control of product quality. The need for tunable and consistent media has thus motivated development of chemically defined media (CDM). However, CDM development strategies are often limited in their screening approaches and unable to reliably assess the impact of media on MSC function, often neglecting high-level interactions of media components such as growth factors. Given that MSC morphology has been shown to predict their immunomodulatory function, we employed a high throughput screening (HTS) approach to elucidate effects of growth factor compositions on MSC phenotype and proliferation in a custom CDM. MethodsHTS of eight growth factors in a chemically defined basal medium (CDBM) was conducted via a two-level, full factorial design using adipose-derived MSCs. Media hits were identified leveraging cell counts and morphological profiles. After validating phenotypic responses to hits across multiple donors, MSCs were cultured over three passages in serum-containing medium (SCM) and CDM hits and assayed for growth and immunomodulatory function. Finally, growth factor concentrations in one hit were further refined, and MSC growth and function was assessed. ResultsOur HTS approach led to the discovery of several CDM formulations that enhanced MSC proliferation and demonstrated wide ranging impacts on MSC immunomodulation. Notably, two hits showed 4X higher growth compared to SCM over 3 passages without compromising immunomodulatory function. Refinement of one CDM hit formulation reduced growth factor concentrations by as much as 90% while maintaining superior growth and similar function to SCM. Altogether, distinct MSC morphological profiles observed from screening were indicative of differential MSC quality that allowed for development of an effective CDM for MSC expansion. ConclusionsOverall, this highlights how our HTS approach led to the development of CDM formulations for robust MSC expansion and serves as a generalizable tool for improvement of MSC manufacturing processes.
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Development of a Robust Consensus Modeling Approach for Identifying Cellular and Media Metabolites Predictive of Mesenchymal Stromal Cell Potency
Abstract Mesenchymal stromal cells (MSCs) have shown promise in regenerative medicine applications due in part to their ability to modulate immune cells. However, MSCs demonstrate significant functional heterogeneity in terms of their immunomodulatory function because of differences in MSC donor/tissue source, as well as non-standardized manufacturing approaches. As MSC metabolism plays a critical role in their ability to expand to therapeutic numbers ex vivo, we comprehensively profiled intracellular and extracellular metabolites throughout the expansion process to identify predictors of immunomodulatory function (T-cell modulation and indoleamine-2,3-dehydrogenase (IDO) activity). Here, we profiled media metabolites in a non-destructive manner through daily sampling and nuclear magnetic resonance (NMR), as well as MSC intracellular metabolites at the end of expansion using mass spectrometry (MS). Using a robust consensus machine learning approach, we were able to identify panels of metabolites predictive of MSC immunomodulatory function for 10 independent MSC lines. This approach consisted of identifying metabolites in 2 or more machine learning models and then building consensus models based on these consensus metabolite panels. Consensus intracellular metabolites with high predictive value included multiple lipid classes (such as phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins) while consensus media metabolites included proline, phenylalanine, and pyruvate. Pathway enrichment identified metabolic pathways significantly associated with MSC function such as sphingolipid signaling and metabolism, arginine and proline metabolism, and autophagy. Overall, this work establishes a generalizable framework for identifying consensus predictive metabolites that predict MSC function, as well as guiding future MSC manufacturing efforts through identification of high-potency MSC lines and metabolic engineering.
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- Award ID(s):
- 1648035
- PAR ID:
- 10442153
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Stem Cells
- Volume:
- 41
- Issue:
- 8
- ISSN:
- 1066-5099
- Format(s):
- Medium: X Size: p. 792-808
- Size(s):
- p. 792-808
- Sponsoring Org:
- National Science Foundation
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