Patent GB-1014712-A (GB)
Title: Improvements in and relating to liquid media in contact with animal cells
IPC: A01N1/02
Filed: 1964-02-27
Abstract: Animal cells are handled, perfused and preserved in a composition comprising a liquid medium compatible with the animal cells, having dissolve therein at least 4% by volume of oxygen. The composition is prepared by contacting the liquid medium with a gaseous atmosphere comprising 80-100% by volume of oxygen, 0-5% by volume of oxygen, 0-5% by volume of carbon dioxide and the balance from 0-20% by volume of gases which are generally non-reactive with the components of the medium and are non-toxic to animal cells, at a total gauge pressure of at least 15 lbs. per sq. inch until at least 4% by volume of oxygen, based on the volume of the liquid medium, is dissolved. Preferably, the liquid medium is agitated for a sufficient time, of the order of 30 minutes, with a gaseous atmosphere comprising 90-95% by volume of oxygen and the balance carbon dioxide at a total gauge pressure between 15 and 30 lbs. per sq. inch.  Non-reactive non-toxic gases mentioned are helium, neon, argon, krypton, xenon, nitrogen, hydrogen and water vapour. Animal cells: whole blood, the erythrocyte and leucocyte fractions of whole blood, bone marrow, and tissue of kidneys, liver, heart, spleen, lungs and the like, obtained from birds, reptiles, fish, rabbits, monkeys, cats, dogs, rodents, horses, and human beings.  The liquid medium may be an isotonic saline solution or a balance salt solution containing 0.8-0.9 weight per cent sodium chloride along with other salts; a solution of an anti-coagulant such as acid-citrate-dextrose, heparin or ethylene-diamine tetra-acetic acid; a solution containing protective additives for reducing cell damage during freezing and thawing such as (i) glycerol, (ii) ethylene glycol, (iii) aldoses and ketoses such as xylose, erythrose, arabinose, ribose, glucose, fructose and galactose, (iv) polysaccharides such as maltose, sucrose, lactose, raffinose and dextran, (v) polyvinyl pyrrolidone, (vi) serum albumin, (vii) dimethyl sulphoxide, (viii) mannitol, (ix) urea, or mixtures of these; and a solution containing nutrients such as amino acids, saccharides, inorganic salts, proteins, vitamins or autologous, homologous or heterologous animal serum. One of several similar specified media includes 2.5 weight per cent glucose, 0.6 weight per cent methylcellulose (15 centipoises viscosity), 20 volume per cent animal serum, 16.9 volume per cent water, 10 volume per cent dimethylsulphoxide, 50 volume per cent Eagle's MEM and 4 volume per cent of dissolved oxygen; others may include egg yolk or a crude soya bean phospholipid fraction consisting essentially of equal proportions of lecithin, cephalin and phosphoinositide. Other specified media are an aqueous solution containing, per 1000 millilitres, 1 g. glucose, 4 g. potassium chloride and 8 g. sodium chloride, Tyrode's medium, and Hank's medium. In examples, the viability of cells of rabbit kidney and rat lung frozen and thawed under carefully controlled conditions (specified) employing compositions according to the invention for the perfusion and suspension of the cells, is compared with that of various control samples of the cells.  The Specification discloses typical procedures for isolating individual cells from thawed cell aggregates and for the preparation of cell cultures.