High Throughput Morphological Screening Identifies Chemically Defined Media that Supports Rapid Mesenchymal Stromal Cell Proliferation

Introduction:: Despite promising preclinical studies, mesenchymal stromal cell (MSC) therapies have yet to achieve clinical translation due in part to their functional heterogeneity and challenges in reproducibly manufacturing consistent MSCs at large scale. Traditional use of undefined media components (i.e. fetal bovine serum (FBS)) to support MSC growth is a major source of heterogeneity. This has motivated the development of chemically defined media (CDM) that include specific recombinant growth factors (GFs) for stimulating cell growth. While CDM formulations for large scale MSC expansion exist, their development is often labor intensive, and many studies dedicated to this effort are limited to investigating only a few CDM formulations with different GF compositions¹. Additionally, many studies consider cell growth as the primary quality metric, whereas a medium’s impact on MSC function is essential to consider for manufacturing clinically relevant cellular products. Therefore, there is a critical need for more comprehensive methods to design CDM that enhances MSC proliferation while maintaining (or enhancing) immunomodulatory function. Morphological profiling using high content imaging has enabled identification of morphological features that predict MSC function². In this study, we applied this high throughput screening (HTS) approach to assess the combinatorial effects of eight different growth factors on MSC proliferation and morphology in a defined basal medium. This screening method allowed for the identification of several media “hits” that increased MSC expansion across multiple passages and produced distinct morphological phenotypes compared to MSCs expanded in serum-containing media (SCM).


Materials and Methods:: Adipose-derived MSCs (RoosterBio) were cultured in traditional SCM or in one of 256 CDM formulations with a fibronectin coating that were defined by a full factorial design wherein combinations of 8 different GF supplements (PeproTech), shown in Table 1A, were tested at two levels each (28 = 256 formulations). After 48 hours, cells were fixed and stained using a modified Cell Painting (CP) protocol (nucleus, actin, Golgi, mitochondria, and endoplasmic reticulum), imaged using Cytation 5 high content imager, and 900 features extracted using a custom CellProfiler pipeline³. "Hits” were identified from cell proliferation and morphological data using multivariate analyses⁴. Subsequently, a follow-up screen including hits and control media (SCM) was conducted using 3 MSC lines from different donors to both validate initial findings from the screen and assess the impact media hits have across different MSC lines. Select hits from the follow-up screen were then chosen to expand MSCs over an extended culture period (3 total passages). Cells expanded in SCM and hit media formulations were analyzed for viability and proliferation at each passage and cryopreserved.


Results, Conclusions, and Discussions:: Results and Discussion: Five GF combinations were identified as hits from the initial and follow up screens (Table 1B). Hits produced distinct MSC morphologies and significantly higher proliferation than the SCM control in both screens. Cells grown in hit media were collectively characterized by smaller area and higher form factor (i.e. more circular, less irregular) than those grown in SCM (Figure 1C). Hits also generated morphologies distinct from each other, such as MSCs in F---PLS- showing more prominent actin filaments and elongation compared to F--I---- (Figure 1C). These properties were conserved during extended culture, and each CDM formulation demonstrated higher fold change in cell growth after passage 1 than the control (Figure 2A). Select hits, namely F--IPLS- and F---PLS-, supported ~9 times greater MSC cumulative growth than SCM after three passages (Figure 2B). These groups also produced ~4 times higher cell yields than FTEIPLSA (Figure 2B), suggesting excessive supplementation of GFs may not be necessary or could even limit MSC proliferation. To our knowledge, the ~1000X cumulative fold change observed for F---PLS- in this study is greater than what other groups who have developed CDM containing similar GFs (i.e. bFGF, IGF, and PDGF) have shown^5,6. It is also of note that many published formulations include more than four GFs, often using seven to eight GFs. This suggests that hits discovered via our HTS platform can support higher MSC proliferation using fewer resources^5,7. While hits identified from the HTS enhance proliferation, immunomodulatory properties of MSCs cultured in these media still need to be assessed.

Conclusion: The results demonstrate that HTS is a generalizable platform that can be used to identify culture conditions to optimize MSC manufacturing. The screen identified five CDM formulations that either outperformed or performed similarly to the SCM control in terms of MSC proliferation over an extended culture period, which showcases the ability for low-cost morphological profiling to aid in more comprehensive and efficient development of CDM. While the CDM formulations identified produce distinct morphological signatures in MSCs, further studies are warranted to understand the relationship between MSC morphology, proliferation, and function.


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