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ORIGINAL ARTICLE Table of Contents   
Year : 2010  |  Volume : 53  |  Issue : 4  |  Page : 742-744
Preservation of continuous cell lines at -85°C: A low-cost alternative for resource limited countries


Department of Virology, Post Graduate Institute of Medical Education and Research, Chandigarh, India

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Date of Web Publication27-Oct-2010
 

   Abstract 

Background: Cell culture is the most popular method of virus propagation because of its high sensitivity. However, the need of high cost liquid nitrogen for storage of cell lines is one of the main limiting factor for its widespread use in developing countries. Objective: The present study was therefore carried out to standardize the preservation of continuous cell lines at deep freezer (-85ºC) for 6 months. Methods: Fixed number of Vero and Hep2 cells were preserved at -85ºC deep freezer in separate vials and were revived at one month interval to check the growth pattern. Results: Both the cell lines could be revived with healthy cells and monolayer was formed within 7-10 days, after storage at -85ºC for 4 months. Conclusion: The present study highlights the utility of -85ºC deep freezer as an alternative to liquid N 2 for preservation of these cell lines at least up to four months.

Keywords: Cell culture, cell preservation, deep freezer, liquid nitrogen

How to cite this article:
Mishra B, Ragini S R, Kashiv I L, Ratho R K. Preservation of continuous cell lines at -85°C: A low-cost alternative for resource limited countries. Indian J Pathol Microbiol 2010;53:742-4

How to cite this URL:
Mishra B, Ragini S R, Kashiv I L, Ratho R K. Preservation of continuous cell lines at -85°C: A low-cost alternative for resource limited countries. Indian J Pathol Microbiol [serial online] 2010 [cited 2019 Dec 15];53:742-4. Available from: http://www.ijpmonline.org/text.asp?2010/53/4/742/72068



   Introduction Top


The importance of virus isolation in viral diagnosis is self explanatory. Considering the regulations imposed on the use of animal host systems and their compromised sensitivity, cell cultures have become an essential component of a routine diagnostic as well as a research oriented virology laboratory. Cell cultures provide a good host system for propagation of majority of clinically important animal viruses. This is essential both for the isolation of the virus from the deeper tissues/organ to growth of the virus for production of viral antigens for various serological and even molecular tests. The noble use of cell culture has been the production of cell cultures based vaccines against life threatening viral infections; Sabin oral polio vaccine, SA 14-14-2 for Japanese encephalitis, rubella, human diploid cell culture vaccine for rabies are among the few. Preservation of cell line is a technically demanding cumbersome process leading to frequent failures in terms of contamination or poor revival rate. The best method available for the long term preservation is storage under liquid N 2 at -196 0 c. A trained technical person with good expertise is essential to handle preservation in liquid nitrogen system. Besides the exuberant running cost, the availability of liquid N 2 , associated health risks like cold burn, asphyxiation, possibility of explosion while removal of vials etc. [1],[2] certainly limits its wide use in the field situation and small laboratories. Therefore, a simple, cost effective and user friendly alternative is essential for routine maintenance of the cell lines and to avoid repeated sub-passage particularly in resource limited countries. Thus, realizing the need, the present study was undertaken to standardize the preservation of commonly used cell lines at -85Ί c deep freezer for a substantial period.


   Materials and Methods Top


Study Design

The experiment was carried out in Vero and Hep 2 cell lines. Both the cell lines were grown in multiple cell culture flasks. After formation of confluent monolayer, cells were collected and fixed number of cells preserved in 12 vials in each batch at -85º c deep freezer. Two vials were revived from each batch at one-month intervals. The percentage of cell adherence, pattern of cell growth, morphology of cells and time taken for monolayer formation were observed and recorded. The process was continued till six months of preservation and was repeated three times with each of Vero and Hep2 cell lines.

Formation of Cell Monolayer

Cells (Vero and Hep 2 ) were grown in 25cm 2 angle neck pre sterile tissue culture flask (Nunclon, Denmark) using Earle's minimum essential media with glutamine and (EMEM; Hi media AT047, India) containing additional glutamine (3%; Sigma, Japan) and fetal bovine serum 8% (Hi media, India) following the standard protocol. [3]

Cryo Preservation of Cells

The basic principle of cell preservation was followed as described by Ian Freshney. [4] After the formation of confluent monolayer, MEM was decanted. Cells were washed twice with MEM. Cell layer was then rinsed with 2 ml of TPVG solution (trypsine 2%, versene 0.02%, and glucose 10%). Final treatment was given with 5 ml of TPVG, kept for 1-2 minutes and then discarded. The detached cells were suspended in MEM. The density of cells were adjusted to 6 X 10 5 /ml. Cell counting was done by the method described by Lennette EH and Schmidt NJ. [3] One ml media containing 6Χ10 [5] cells were then distributed to each cryo vial; 12 such vials were prepared in each batch. To this cell suspension 10% Di methyl Sulphoxide (DMSO) (Amresco, USA) was added drop wise with continuous swirling, followed by 20% fetal bovine serum. All the cryo vials were labeled for cell type, passage number and date of preservation.

Storage of Cells

All the cryo vials were first stored for two hours at 4ºC. The exact temperature was achieved by keeping the vials in the inner side of the refrigerator below the freezing chamber. The temperature was ensured by measuring with digital thermometer. Then the vials were shifted in an ice box and kept at -20ºC for two hours, after which vials were finally stored at -85ºC deep freezer.

Revival of Cell

The procedure for cell revival was followed as per the standard protocol with minor modifications. [3] Two vials were revived every month, at one month interval till six months of preservation. Cells were brought to 37ºC by rapidly thawing in 37ºC water bath. Cells were then slowly centrifuged at 500-700 rpm for 10 minutes. Supernatant was discarded and cells were suspended with pre cooled MEM. Viable cell count was made at this stage and all the cells were seeded in each tissue culture flask of 25cm 2 containing cold MEM and 10% FBS and incubated at 37ºC. After 24 hours, cells were replaced with fresh MEM and FBS. Cells were then observed every day to note the pattern of cell growth, morphology, adherence pattern and their rate of growth in terms of time taken for the formation of confluent monolayer. The percentage of cells showing granularity was graded as + to ++++ (25% to 100%). The observations were recorded separately by two experienced virologists having at least 10 years of experience in tissue culture.


   Results Top


Both Vero and Hep2 cell lines preserved up to three months at -85ºC could be revived with healthy cells and cell monolayer was formed within one week [Figure 1], while after four months, it took more than one week for monolayer formation with minimum granularity. The details regarding the number of viable cells revived, pattern of growth, percentage of cell adherence and time taken to form monolayer are given in [Table 1].
Table 1 :Growth status after revival from 1-6 months of cells preserved at -85ºC


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Figure 1 :Vero cell confluent monolayer after storage at -85°C for 4 months

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   Discussion and Conclusion Top


Preservation of structurally intact living cells and tissues at very low temperature is known as cryopreservation. The biological effects of cooling are governed by the freezing of water, which in turn leads to concentration of various solutes present in the remaining liquid phase. Formation of ice crystal inside the cell destroys it by causing direct mechanical injury or indirectly due to the changes in the liquid phase composition. [5] Therefore optimal freezing of cells for maximum recovery of viable cells on thawing depends on two basic factors; i) minimizing the intracellular crystal formation, and ii) reducing cell damage from high concentration of solutes that is formed due to freezing of intra cellular water. [4],[6],[7] This is achieved by slow cooling, using hydrophilic cryoprotectant, storing the cell at lowest possible temperature, and rapid thawing. [4]

Preservation of common cell lines at -85ºC temperature is used in established tissue culture laboratory; however, due to the lack of systematically conducted published study, the method is still not widely used. The present study was therefore conducted over a period of 6 months to look for the maximal recovery of cells when stored at -85ºC temperature for different time period.

Vero and Hep2 cell lines are amongst the commonly used continuous cell lines in the tissue culture laboratory and used in combination for the isolation of commonly encountered clinical pathogens like HSV1, HSV2, measles, enteroviruses, adenoviruses. The preservation of cell lines is important particularly to maintain the lowest number of passage for a longer time, in order to preserve the genetic stability and support the growth of virus to express proper viral antigens. Cell lines as well as infected cell cultures are processed in liquid N 2 (-196ºC) as a customary. But the procedure demands good expertise and timely handling of the cell lines before putting inside the liquid N 2 .

Handling of liquid N 2 can even lead to serious hazards like cold burn and asphyxiation. [1] Moreover, the regular requirement of refilling of liquid N2 because of its evaporative nature makes it more cumbersome and expensive. The successful preservation of commonly used cell lines for 3-4 months at -85ºC deep freezer in the present study has proved it as a cost effective and user friendly alternative to liquid N2 as it involves one time investment. Besides this, -85ºC deep freezer also fulfills several other requirements of a microbiology laboratory like; storage of serum and other clinical samples, molecular reagents, antigens and other chemicals. Adaptation of this method of cell preservation facilitates the viral works at most of the microbiology laboratories where, in addition to the cost factor, availability of liquid N 2 supply is the main hindrance.

Isolation of viruses like Herpes simplex virus 1 (HSV1) and adenovirus have been done in the stored cells (after four months) and confirmed by specific polymerase chain reactions (PCR) and monoclonal antibody. The increased granularity with cell clone formation and requirement of prolonged period beyond 1 week time for a near total mono layer formation however indicates the non-suitability of those cells for isolation purpose.

The present cell preservation system needs only common laboratory instruments like -20ºC deep freezer and -85ºC deep freezers which are available in most of the microbiology laboratories. Thus delaying a sub culture up to three to four months obviates the higher passage levels and reduces the running cost of maintenance of cell lines thereby encouraging its widespread use in microbiology laboratories having these facilities.

 
   References Top

1.Tomlinson M. Risk management in cryopreservation associated with assisted reproduction. Cryo Lett 2008;29:165-74.  Back to cited text no. 1
    
2.Wyatt TD. Overcoming the hazards-of storing cultures in liquid nitrogen. J Clin Pathol 1984;37:840.  Back to cited text no. 2
[PUBMED]  [FULLTEXT]  
3.Lennette EH, Schmidt NJ. Diagnostic procedures for viral and rickettsial infections, 5th ed. Washington: American Public Health Association; 1979.  Back to cited text no. 3
    
4.Ian FR. Culture of animal cells. A manual of basic technique, 5th ed. Wiley-Leiss; 2005.  Back to cited text no. 4
    
5.Pegg DE. Principles of cryopreservation. Methods Mol Biol 2007;368:39-57.  Back to cited text no. 5
[PUBMED]    
6.Batista Lobo S, Denyer M, Gopalan RC, Javid FA. Technique for cryopreservation of intestinal smooth muscle cells. Cryobiology 2008;57:186-9.   Back to cited text no. 6
[PUBMED]  [FULLTEXT]  
7.Rubinsky B, Perez PA, Carlson ME. The thermodynamic principles of isochoric cryopreservation. Cryobiology 2005;50:121-38.  Back to cited text no. 7
[PUBMED]  [FULLTEXT]  

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Correspondence Address:
R K Ratho
Department of Virology, Post Graduate Institute of Medical Education and Research, Chandigarh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.72068

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    Figures

  [Figure 1]
 
 
    Tables

  [Table 1]

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