In the 4th Session of "Panda Class – CGT Series Lectures"

We invited Dr. Chen Min, R&D Director of BioEngine, to deliver a themed presentation titled Strategies and Technical Challenges for Efficient Mesenchymal Stem Cell Culture. The presentation elaborated on the key points and strategies for the efficient culture of mesenchymal stem cells (MSCs). During the interactive Q&A session, participants actively raised a wide range of questions. Dr. Chen Min provided thorough and detailed answers to most of them. Unfortunately, due to time constraints, not all questions could be addressed. To this end, we have specially compiled and organized the questions raised during the live broadcast.

Q1: What are the differences in cell culture practices for MSCs derived from different sources?

A1: There are no significant differences in the operation procedures of cell culture. However, it should be noted that MSCs from different sources exhibit certain variations in surface markers and their potential for differentiation into various lineages.

Q2: What are the causes of large batch-to-batch variations in exosome production?

A2: a. Variations are introduced during the cell expansion and passage stage of cell culture; b. Variations stem from differences in cell status at the time of seeding during the exosome production stage; c. Exogenous exosomes are introduced during the processes of cell culture and exosome expression; d. Variations are caused by small-scale or discontinuous operations for exosome collection and purification.

Q3: Should the cells be round after complete digestion? Is it normal if the cell borders appear polygonal?

A3: During MSC passage, MSCs should assume a round shape after complete digestion. The presence of distinct borders or a polygonal morphology may indicate abnormal cell growth.

Q4: Do I need to purchase adhesion factors separately for CD media? What concentration should be used?

A4: Adhesion factors need to be purchased independently. It is recommended to use the concentration suggested by the supplier or determine the optimal concentration through gradient experiments.

Q5: How do the proliferation capacity and stemness of MSCs compare when cultured in chemically defined (CD) media versus xeno-free media?

A5: According to the internal test results of BioEngine's R&D team, the expansion of MSCs using BioEngine's Omni CD MSC Medium does not exert any adverse effects on the growth, stemness, or other performance attributes of MSCs.

Q6: Could you elaborate on the procedures and precautions for cell isolation and cryopreservation, as well as the formulation of cryopreservation medium?

A6: The isolation methods for MSCs vary depending on their tissue sources. For example, density gradient centrifugation is commonly used for isolating bone marrow-derived MSCs; the explant outgrowth method is typically applied for umbilical cord-derived MSCs; type I collagenase digestion is often employed for adipose-derived MSCs; and the combined use of trypsin and type II collagenase is suitable for digesting placental amniotic tissue to obtain MSCs.

Precautions for cell isolation operations:

Biological samples should be kept fresh. Minimize the time interval between sample collection and isolation, and store or transport the samples at low temperature.

It is advisable to add an appropriate concentration of antibiotics to the reagents and medium during the cell isolation process. Conduct sterility testing by sampling a small amount of supernatant shortly before plating-such as after cell sieving or the final centrifugation and resuspension step. Strictly follow aseptic operation guidelines throughout the isolation process.

Cryopreserve cells at early passages as much as possible, and carry out cell identification work simultaneously.

Cell cryopreservation:The cryopreservation procedure is essentially the same as that for other cell types, following the principle of "slow freezing". After harvesting the cells and resuspending them in the cryopreservation medium, the cooling rate must be controlled to be slow.The cryopreservation medium can be prepared by adding 5–10% DMSO to the medium used for cell passage, or commercial cryopreservation medium products can be directly adopted.

Q7: How to terminate the action of collagenase during the isolation of adipose-derived MSCs?

A7: After adding collagenase to complete the digestion step for isolating adipose-derived MSCs, the enzyme should be removed by washing the cells with PBS or culture medium followed by centrifugation. It is not recommended to use collagenase for routine MSC passage, as it may cause damage to the cells.

Q8: How to address the individual differences in MSCs derived from umbilical cord or adipose tissue?

A8: Differences between tissues from different donors are inherent and cannot be completely eliminated. However, efforts can be made to minimize these variations by: selecting donors with similar ages and health statuses; standardizing the processes of biological sample collection and cell isolation; and establishing standardized criteria for evaluating the growth status and identification results of the isolated cells, thereby avoiding introducing additional variations caused by inconsistent operations.