Factors affecting cell culture growth

 

 

DDx no growth or poor growth

 

 

 

·         Sterilization

·         Contamination

·         Particularly a problem with autopsy and abortus tissues

·         Antimicrobials added to collection media and culture media

·         Collagenase dispersal helps reduce contamination in tissues

·         Incubators are a frequent source

1.    Frequent cleaning / decontamination

·         Tissue handling:

·         Freezing

·         Formalin fixation

·         Sterile saline is best

·         Time factor involved in handling of specimens is directly correlated with success of culture growth (Kyle et al, 1996)

·         Collagenase dispersal

·         Improves speed of growth

·         Inoculation density – human monolayer cells grow poorly if inoculated at too low or too high a density

·         Sample too large

·         Media:

·         Water used

1.    Tap water is poor

2.    pH near neutral (not acidic)

·         15% bovine serum (5-30%)

1.    Different lots of serum may vary in their performance – need to test each new lot

2.    Prescreened for mycoplasma

·         temperature:

·         37-37.5oC

·         pH:

·         7.2 – 7.4

·         Bicarbonate buffer helps maintain the pH

·         CO2 in air helps maintain the pH

·         Phenol red in the solution helps indicate the pH

1.    Pink-orange is the proper pH

2.    Yellow is too acidic

3.    Pink is too alkaline

·         Humidity:

·         Close to saturation

·         Frequent opening / closing of incubator may alter the above

·         Age of culture:

·         Limit of 50-60 population doublings for human diploid cells

1.    Can be extended if cells are frozen in liquid nitrogen early on

1.    Controlled rate (1oC/min)

 

References:

·         Barch MJ, Knutsen T, Spurbeck JL. The AGT Cytogenetics Laboratory Manual. 3rd ed. Lippincott Williams & Wilkins; 1997.

·         Winsor EJT, Windrim R, Chitayat D, et al. Success rate for culture of fetal postmortem tissue is dependent on the method of pregnancy termination. Fetal Diagn Ther. 2005;20(4):306-8.

·         Bernick BA, Ufberg DD, Nemiroff R, Donnenfeld A, Tolosa JE. Success rate of cytogenetic analysis at the time of second-trimester dilation and evacuation. Am J Obstet Gynecol. 1998;179(4):957-61.

·         Rodgers CS, Creasy MR, Fitchett M, et al. Solid tissue culture for cytogenetic analysis: a collaborative survey for the Association of Clinical Cytogeneticists. J Clin Pathol. 1996;49(8):638-41.

·         Kyle PM, Sepulveda W, Blunt S, et al. High failure rate of postmortem karyotyping after termination for fetal abnormality. Obstet Gynecol. 1996;88(5):859-62.