Cryopreservation is an essential cell culture maintenance activity in biomedical labs to preserve cells for future use. There are various aspects of cryopreservation which need to be learnt and properly performed so as to obtain a good viable population of cells after thawing, in case a culture follows. Keeping these things in mind, we had an expert session with Dr. Sachin Kadam on the Do’s and Don’ts of cryopreservation.
Dr. SachinKadam, with over 13 years of experience in stem cell research, has fueled Kosheeka’s research and development with his esteemed expertise and scientific zeal. In this article, we put forward a short piece on our Q&A session about cryopreservation techniques and ethics.
Which type of threaded cryovials are preferred by cell culture researchers: internal or external?
Some researchers prefer external threaded cryovials so that no outside particles or contaminants get in the vial, thus making it safer for cryopreservation. Whereas, some researchers choose internal threaded vials as they fit better in the canisters and freezer boxes.
DMSO is used as a cryopreserving agent. What alternatives would you suggest for primary cell culture applications?
DMSO is an intracellular cryoprotective agent and many alternatives have been reported for tissue freezing, including non-penetrating cryoprotectants like glucose, sucrose, galactose, or trehalose, and intracellular cryoprotectants like ethylene glycol, propylene glycol, glycerol, formamide, methanol, and butanediol. Researchers generally use freezing media with 10% DMSO and FBS for cell culture applications but the use of polyvinylpyrrolidone (PVP) has been suggested in some literature reports as alternative to DMSO.
Which points in the cryopreservation protocols should be primarily focused on?
There are some main points to focus while going ahead with cryopreservation. Firstly, freezing good quality cells is an important criteria. Researchers should be aware that high viability is not related with high population. Cells should be frozen after being passaged for 2-3 days with less than 90 percent confluency. A population of 2 x 10^6 cells/ml is mostly the typical cryopreservation density. Secondly, using DMSO as a cryoprotecting agent along with FBS or Ficoll to the freezing medium. In this regard, researchers should use fresh reagents for freezing procedure to gain high recovery efficiency. A constant rate of freezing at -1°C/min is suited for cell culture applications. Moreover, keeping cryovials in the vapor phase between -140°C and -180°C reduces the risk of leaky vials or unexpected damage. Last but not the least, cells should be thawed rapidly by placing the cryovials in a 37°C water bath to prevent osmotic shock.
Is there any way to store cryovials if liquid nitrogen availability is an issue?
A temperature of -80°C might be a short-term alternative to store primary cells but with the use of 10% Ficoll 70 to the 10% DMSO in the freezing medium can help in enhancing recovery viability.
We thank Dr. Sachin Kadam for his valuable insights on cryopreservation for primary cell culture and a lot of troubleshooting still awaits for various other crucial points in the process of good cell culture practice. For any information on cryopreservation or primary cell culture practices, consult Kosheeka at email@example.com