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How To Overcome The Challenges In Cryopreservation?

Cryopreservation has become one of the major steps to focus on enhancing commercial and clinical cellular experiment-based cell culture process. Non-optimized substandard protocols and reagents used in cell culture can lead to damage of cells and lower cell viability. Besides that, the lack of optimized protocols in cell culture practice might also lead to decreased cell functionality, batch variation, genetic variations, and the epigenetic difference in cell subpopulations. In the case of the cryopreservation process in the cell culture process, the major focus lies on freezing and thawing techniques. Secondly, a proper established protocol of preparing freezing media also matters in terms of maintaining the cell viability of the cryopreserved cells. Aside from that, thawing and revival of the cells involve a great deal of good practice and optimization of centrifugation and media changing techniques. 

The Common Slip-Ups Of Cryopreservation

Every single step in the whole process of freezing and thawing can lead to a number of sillies over looked mistakes but if taken care of, a simple fix can go a long way to make your cell culture more efficient. 


Freeze only the best quality cells. Common points to remember based on poor culture practice are high cell passage numbers, highly confluent cells, microbial contamination presence, cultures having residual DMSO or trypsin, rigorous centrifugation for cell pelleting, rigorous pipetting for resuspension of cells, etc. Avoid all these mistakes in your cell culture before deciding upon freezing your cells and always remember to freeze your cells at early passage numbers. 


Freezing cells should start at 4° C before adding cryopreserving and freezing should start as soon as the freezing medium is added. Adding the freezing medium too early or too late will again cause viability problems in your cells due to undesirable ice formation, excessive cell dehydration, and subsequent cell death. Besides that, the proper concentration of freezing media and cells should be used for maintaining optimal cell viability after storage.


The positive benefits of cryopreservation can be only witnessed if the cells are stored properly and this involves knowing whether your cells require storing in the vapor phase or liquid phase of liquid nitrogen canister. Temperatures below -130° C are required to optimally protect the cell viability culture.


The primary issue in this step is thawing the cells too slowly and allowing the vial contents to warm before centrifugation and removing the cryoprotectant. Always ensure that the vial contents are diluted with fresh growth media before reaching room temperatures.

Critically Advanced Challenges in Cryopreservation

Developments in the cell culture process have started influencing the advancements in cryopreservation techniques and this includes the usage of serum-free, maybe chemically defined, media as freezing media without DMSO. Residual DMSO has been challenges in cryopreservation in cell culture as it proves toxic to the cells and therefore, using alternate GMP compliant formulations might prove less potentially harmful for the cell culture. The major issue of DMSO in cell culture is in the case of cell therapy where DMSO is a part of the therapeutic formulation.

To date, although there have been sincere scientific attempts to replace DMSO, it remains the standard with protocols involving lower concentrations (less than 10%) and intensive washing steps. Some scientists have also researched upon newer DMSO-free cooling methods using techniques of vitrification or nanocrystallization. But to date, DMSO-based cryopreservation has been deemed most cost-effective and besides lowering its concentration, researchers also recommend keeping a high cell concentration during cryopreservation in order to have a reduced absolute DMSO volume in the therapeutic formulations.

Moreover, although DMSO-alternative discussions of vitrification and devitrification have been popular in cryopreservation research, these processes in 3D tissues or organoids might be damaging for the cells and thus in order to evade the problem of crystal formation, some researchers are also exploring hydrogel systems as conduits for cryopreservation. 

As the cell culture process becomes more complex, for example, high throughput screening plates for drug testing, cryopreservation also becomes more challenging. There are increasing opportunities for using cryopreservation of the high throughput screening plates for drug screening and drug toxicity applications but the challenges in cryopreservation is maintaining the batch-to-batch consistency of these plates. In this regard, using a cryopreserved batch of the same donor tissue high screening throughput plates is the optimal way to ensure such experimental consistency for drug testing or screening. Thus, research on improving such techniques might be a challenging future endeavor for enhanced cell culture flow. 

Are you a cell culture researcher? Is your lab looking forward to procuring the best quality of tissue-specific and species-specific primary cell? Contact Kosheeka today at for all your related inquiries.

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