The use of pronase in tissue culture: A comparison with trypsin

Abstract

Pronase is a proteolytic enzyme recently introduced as an effective dispersing agent of tissue culture cells. We have found it to be a most rapid and complete dispersing agent for primary fibroblastic cell lines and clearly superior to trypsin. O n the other hand, with certain continuous epithelial cell lines its completeness of dispersion is inferior to that of trypsin necessitating study of each cell line individually with both enzymes to select the most advantageous one.Since Gwatkin and Thompson ('64) pointed out advantages of dispersing cells in vitro with the crude proteolytic enzyme preparation, pronase, considerable differences have been expressed on its value. Kahn, Ashwood-Smith and Robinson ('65) have noted failure of their cell lines to survive following dispersion with pronase. On the other hand, Sullivan and Schafer ('66) have used the enzyme over approximately two years without difficulty and feel it has been the single most satisfactory enzyme used in their laboratory for producing single cell suspensions without clumps. Weinstein ('66) has confirmed Gwatkin and Thompson's original observation finding that the enzyme more rapidly disperses diploid fibroblastic cells and more effectively eliminates clumping than trypsin. He also showed that there was no significant effect on the number of cell divisions of a diploid fibroblastic strain nor on the chromosome morphology.Our five year's experience with pronase as a cell dispersing agent is contained in the ensuing manuscript. MATERIALS AND METHODSCell lines. Continuous: HeLa cell line 299 and KB cells (obtained from Microbiological Associates). Monkey kidney epithelium was obtained from Dr. George Dubes of the Department of Microbiology.The fibroblastic cell lines were grown from aseptically collected surgical specimens which were minced into 1 mm cubes and placed in growth medium. Primary.J. CELL. PHYSIOL., 75: 159-162. M e d i u m .HeLa cells were grown in a modified Eagle's basal medium (Treadwell and Ross, '62) containing 10% human serum and 10% fetal calf serum.KBM cells were grown in the above medium with 10% calf serum.The primary cultures were grown in a modified medium 199 (Treadwell and Ross, '62) containing 10% human serum and 10% fetal calf serum.Enzymes. Trypsin (Nutritional Biochemical Company, 1 : 300) was made up as a 2% solution in sterile balanced salt solution and stored in aliquots at -20" C.Pronase (Cal. Biochem. Co. B-Grade) was made up in a balanced salt solution at 1% concentration and stored at -20" C until used. Once thawed the enzyme is stored at 4" C and used over a one to two week period without loss of noticeable dispersing potency. Subculture m e t h o d sThe continuous cultures were routinely passed by draining growth medium and adding a balanced salt solution containing 0.2% trypsin for 20 minutes at 36.5" C . Then the cells were gently dispersed by pipetting six times followed by centrifugation at 60 X g for six minutes. The cell pellet was then redispersed in growth medium after discarding the supernate. The ...

Development of viral nanoparticles for efficient intracellular delivery

“…The ratio of surface-bound and internalized VNPs was determined by treating cells previously exposed to CPMV and CPMV–R5 formulations with the enzyme pronase (a proteolytic enzyme and alternative to trypsin 17 ), to remove particles bound to the external membrane surface. The fluorescence signal from treated and untreated samples was comparable in both formulations, suggesting that CPMV nanoparticles and R5-conjugates were internalized.…”

Regional Effects of Enzymatic Digestion on Knee Meniscus Cell Yield and Phenotype for Tissue Engineering

“…In agreement with the present findings, previous reports comparing the efficacy of trypsin and pronase have reported more favorable tissue digestion with pronase, with digestion occurring more rapidly and the resulting cell solution containing fewer cell clumps. 42,43 As pronase is known to act on a wide variety of substrates, breaking proteins down into their individual amino acids, it is likely that pretreatment of meniscus tissue with this enzyme increases tissue permeability and allows the following collagenase enzyme better access to collagen molecules throughout the tissue. The high cell yield resulting from the P/C isolation protocol represents a vast improvement over other common digestion methods, and maybe applicable to other fibrocartilaginous or fibrous tissues such as the temporomandibular joint disc or tendons.…”

Separation of Gastric Mucosal Cells of Rat with Proteolytic Enzymes, Pronase and Trypsin, with Special Reference to the Collection, Morphology and Viability of the Generative Cells

“…They applied the techniques used in the exfoliative cytology of the stomach, using proteolytic enzymes, trypsin and pancreatin. We employed pronase in this experiment which has been reported to have a much higher ability than any other proteolytic enzymes to disaggregate cells for primary culture as well as for subcultivation (Gwatkin and Thomson 1964;Weinstein 1966;Foley and Aftonomos 1970). The ultimate purpose of our works is to establish cell lines from gastric mucosal cells, so that the interest was naturally focused on the separation of cells with growth potential, i.e.…”