Brief Introduction to How Mesenchymal Stem Cells and Their Derivatives are Bioprocessed

3.    Bioprocess Development for Secretome Derived Products

Requirements:

The following are the requirements of the bioprocessing of the MSC secretome and associated EVs in order to properly characterize and access its therapeutic potential:

1. Standardization of the expansion of MSC culture, collection of secretome and isolation of defined components.
2. Expansion of the MSC population in vitro using defined cultural conditions which are reproducible, scalable and well-controlled will help in limiting heterogeneity and enhancing predictability in the composition and function of secretome derived products.
3. Development of defined protocols for storage, transport and delivery of secretome derived products.

Challenges:

The challenges of bioprocessing MSC secretome for therapeutic applications are summarized as follows:

1. Currently, no reliable assay exists that can test EV membrane integrity, which may impact the therapeutic benefits or reproducibility.
2. No standardized or quantified list of the secretome components is available.
3. There is a need to look for active molecules in the secretome that might be oncogenic, as they might potentially alter the genome of the recipient cell and pose the risk of developing cancer.
4. Despite having the potential for therapeutic applications in various cancer treatments, there is evidence that certain MSC phenotypes may contribute to tumour progression and metastasis. Hence, the oncogenic potential of MSC and MSC secretome makes their application controversial.

Steps:

a.      Deciding/Formulating Culture Medium

For characterizing and analyzing the secretome, a sound understanding of the media composition is very crucial. Majority of studies that culture MSCs report usage of Fetal Bovine Serum (FBS) since it has a low amount of antibodies and a high amount of growth factors.

Concerns:

• FBS has a highly variable composition. It generally depends on when, where and how it was collected.
• FBS might also contain contamination of animal-derived infectious agents. They might be retained within the MSCs. If this is the case, there might be an immunological response when the cells or the cell products are transplanted to a patient.

Solution:

• The serum’s exosome must be removed to prevent mixing up with those derived from MSCs.
• Alternatives of FBS include human platelet lysate (HPL) supplementation in media and a variety of chemically defined serum-free media (SFM). Some of the research labs have also developed their own xeno- or serum-free media.

b.      Isolating MSCs

MSCs can be obtained from tissue sources that preserve the potential to regenerate and differentiate. Some of these tissues include the umbilical cord, cord blood, adipose tissue, and bone marrow. In-depth protocols for isolating MSCs from each of these sources can be found here.

Concerns:

• MSCs have a certain degree of commitment in terms of their fate. So, they can vary in function depending on the tissue they are isolated from. Accordingly, they also display a distinct secretome profile specific to their native tissue.

Solution:

• It is imperative to match donor characteristics and tissue source to secretome functionality in the respective disease models.
• MSCs can also be genetically engineered for enhancing their therapeutic benefits and tailoring their applications with respect to a specific treatment.

c.      Culture Condition Optimization and Population Expansion

It is vital to match the culture medium, condition and source, to the intended application of the MSCs as the culture condition can impact the composition and bioactivity of the secretome and its derivatives. Culture conditions need to be varied in small batches and in defined time points to conduct small-scale studies of the variabilities in the MSC population and obtain the optimal culture condition.

Once the optimal conditions are obtained, T-flasks can be used to expand the MSC population.

Concerns:

• If we use T-flask for culture expansion there can be various challenges. The production in T-flask can only be done in batches, which might result in huge variability in the cell populations and their products.

Solution:

• Suspension Bioreactors can be used for the high production of MSC-derived products. Here, the cells grow in suspension in the presence of mechanical agitation. This is useful in obtaining homogeneous productivity in batches.
• Mechanical stimuli have also been found to enhance the chondrogenic profile similarity amongst the batches.
• Also, another emerging process is the 3-D Solenoid Culture, here the MSCs grow as 3-D aggregates. They attach to themselves rather than to the surface of the culture container. This is based on hanging-drop method and exhibits enhanced anti-inflammatory, angiogenic and regeneration properties. The culture situation also triggers the mechano-physical properties of the new population od MSCs with differed gene expressions.

d.      Isolation of MSC Secretome, Storage and Delivery

Depending on the culture growth phase, the type and amount of the MSC-derived products vary. This needs to be adequately determined, standardized and monitored to obtain the desired product in optimal amount. MSC-derived secretome can then be isolated by ultracentrifugation, microfluid approach, anion exchange, or, polyethylene glycol−based precipitation etc.

Once the secretome is isolated, it needs to be stored before it finds its therapeutic or scientific applications. Therefore, it is important to consider the effects of freeze-thaw, stability over different ranges of temperatures, and the effects of freeze-drying components in order to preserve the biological activity of the isolated products. Research shows that cells at -20C can be stored for long period without any trace of chemical changes within it.

A cell-free approach of delivery in relation to exosomes has great demands in medicine and immunomodulation, but the problem lies in cell retention. Usage of photoinduced imine-cross-linking hydrogels to embed the MSCs and their derived products has shed some light in this regard. An appreciable extent of retention of drugs was observed when injected with Matrigel Scaffold.

Despite the application of hydrogels in delivering MSCs and MSC-derived products, there still remains the necessity of further studies to formulate the best delivery strategy that would eventually solve the hurdles of Stem Cell Therapy.

By,

Shruti Mandal (2nd-year BS-MS student, IISER Kolkata)

Edited by,

Diptatanu Das (5th-year BS-MS student, Department of Biological Sciences, IISER Kolkata)

References:

• Phelps, J., Sanati-Nezhad, A., Ungrin, M., Duncan, N. A., & Sen, A. (2018). Bioprocessing of Mesenchymal Stem Cells and Their Derivatives: Toward Cell-Free Therapeutics. Stem Cells International, 2018, 1–23. doi: 10.1155/2018/9415367 
• Börger, V., Staubach, S., Dittrich, R., Stambouli, O., & Giebel, B. (2020). Scaled isolation of mesenchymal stem/stromal cell-derived extracellular vesicles. Current Protocols in Stem Cell Biology, 55, e128. doi: 10.1002/cpsc.128
• Liu X, Yang Y, Li Y, Niu X, Zhao B, Wang Y, Bao C, Xie Z, Lin Q, Zhu L. Integration of stem cell-derived exosomes with in situ hydrogel glue as a promising tissue patch for articular cartilage regeneration. Nanoscale. 2017 Mar 30;9(13):4430-4438. doi: 10.1039/c7nr00352h.
• Cover Image adapted from oxfordscience.org

About the author

Shruti Mandal, a sophomore of IISER Kolkata is ongoing her BS-MS course, in the department of biological sciences. She likes to introduce herself as a – ‘Jack of all trades, master of none ‘. She is a regular writer at The Qrius Rhino and you can find all her blogs here.