# US 5,700,632 A · General method to quickly remove cryoprotectants from animal cells while maintaining viability > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,700,632 A - Document ID: US5700632A - Status: Granted - Kind: A - Application number: 08/474,477 - Filed: 1995-06-07 - Granted: 1997-12-23 - Earliest claimed: 1994-05-27 - Assignees: Methodist Hospital of Indiana, Inc. - Inventors: John K. Critser; D. Y. Gao - Web page: https://patentgrep.com/patent/US5700632A ## CPC - A01N 1/146: Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving - A01N 1/125: Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators - A61K 35/52: Sperm; Prostate; Seminal fluid; Leydig cells of testes - A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract A mathematical model, and membranes and devices based upon that model, to optimize protocols for the addition or removal of cryoprotectant to or from biological cells. This disclosure describes the use of four equations to predict optimal protocols to add or remove cryoprotectant to or from biological cells. The equations particularly require experimentally found data, specific to cell-type and species, regarding the osmotic tolerance of the cells, where osmotic tolerance refers to the cells ability to shrink or swell to various changes in osmolality without injury. The equations further require the cryoprotectant permeability coefficient and the water permeability coefficient of the particular cells' plasma, membrane. These coefficients are found with experimental data of the knetic volume change of the cell-type to a known concentration and temperature of cryoprotectant. Also disclosed are particularly preferred methods and devices to add or remove cryoprotectant to or from these cells based upon these equations. ## Claims ### Claim 1 A method to remove cryoprotectant from the cells of an animal species containing cryoprotectant comprising: a) predetermining an upper cellular volume limit above which a user-defined fraction of cells lose their viability, b) selecting a first concentration of cryoprotectant solution which is lower than that within the cells, c) calculating whether said first concentration of cryoprotectant solution will cause the cells to exceed the upper cellular volume limit by a calculation which uses the water permeability coefficient and the cryoprotectant permeability coefficient of the cells, repeating steps b) and c) as necessary in order to calculate a predetermined concentration of cryoprotectant solution which will permit the decrease of the concentration of cryoprotectant in the cells while still maintaining the viability of a user-defined fraction of the cells, d) contacting said cells containing cryoprotectant with the solution of the predetermined concentration of cryoprotectant. ### Claim 2 (depends on 1) The method of claim 1, wherein said upper cellular volume limit is predetermined by swelling said cells of said animal species in increasingly hypotonic conditions until a user-defined fraction of said cells lose their viability. ### Claim 3 (depends on 2) The method of claim 2, wherein said cells are sperm cells and said viability is measured by analysis of sperm motility. ### Claim 4 (depends on 1) The method of claim 1, wherein said cryoprotectant comprises glycerol. ### Claim 5 (depends on 1) The method of claim 1, wherein said contacting comprises contacting said cells with the cryoprotectant solution is in two or more substantially constant volumes. ### Claim 6 (depends on 1) The method of claim 1, wherein said contacting comprises contacting said cells with the cryoprotectant solution in two or more volumes wherein said volumes reduce the level of cryoprotectant in the cells by substantially constant decrements in molarity. ### Claim 7 (depends on 1) The method of claim 1, wherein said cells are sperm cells and said upper cellular volume limit is about 110% of the isotonic volume of said sperm cells. ### Claim 8 (depends on 1) The method of claim 1, wherein said cells are blood cells. ### Claim 9 (depends on 1) The method of claim 1, wherein said cells are pancreatic islet cells. ### Claim 10 (depends on 1) The method of claim 1, wherein steps (b), (c) and (d) are repeated for a second concentration of cryoprotectant that is more dilute than said first concentration. ### Claim 11 A method to remove cryoprotectant from the cells of an animal species containing cryoprotectant comprising: a) predetermining an upper cellular volume limit above which a user-defined fraction of cells lose their viability, b) selecting a first rate of cryoprotectant removal, c) calculating whether said first rate of cryoprotectant removal will cause the cells to exceed the upper cellular volume limit by a calculation which uses the water permeability coefficient and the cryoprotectant permeability coefficient of the cells, repeating steps b) and c) as necessary in order to calculate a predetermined rate of cryoprotectant solution removal while still maintaining the viability of a user-defined fraction of the cells, d) contacting said cells containing cryoprotectant with a first solution of cryoprotectant and continually lowering the cryoprotectant concentration in the solution at the predetermined rate. ### Claim 12 (depends on 11) The method of claim 11, wherein said upper cellular volume limit is predetermined by swelling said cells of said animal species in increasingly hypotonic conditions until a predetermined fraction of said cells lose their viability. ### Claim 13 (depends on 12) The method of claim 12, wherein said cells are sperm cells and said viability is measured by analysis of sperm motility. ### Claim 14 (depends on 11) The method of claim 11, wherein said cryoprotectant is selected from the group consisting of dimethyl sulfoxide, glycerol, and ethylene glycol. ### Claim 15 (depends on 11) The method of claim 11, wherein said continually lowering step is performed with a porous membrane that separates said first solution and a second solution, said membrane regulating the the flow of cryoprotectant and water between said first and second solutions. ### Claim 16 (depends on 15) The method of claim 15, wherein said second solution begins with a cryoprotectant concentration that is lower than the cryoprotectant concentration of the first solution. ### Claim 17 (depends on 11) The method of claim 11, wherein said cells are blood cells. ### Claim 18 (depends on 11) The method of claim 11, wherein said cells are pancreatic islet cells. ### Claim 19 A method to remove cryoprotectant from the cells of an animal species containing cryoprotectant, comprising: contacting said cells containing cryoprotectant with one or more stepwise additions of aliquots containing said cryoprotectant, wherein each aliquot has a predetermined concentration of said cryoprotectant that is lower than the cryoprotectant concentration within the cells with which it is contacted, said predetermined concentration substantially preventing the volumetric excursion of said cells beyond a predetermined upper cellular volume limit, and said predetermined concentration being predetermined by determining the cellular volume of said animal cells for a given concentration of said cryoprotectant with differential equations: ##EQU5## wherein J.sub.v =total volume flux, V=cell volume, t=time, N=mole number of the solute, A.sub.c =cell surface area, L.sub.p =water permeability coefficient, C=concentration of solute, J.sub.CPA =cryoprotectant flux across the cell membrane, superscript e=extracellular, superscript i=intracellular, C.sub.CPA =average cryoprotectant concentration of extracellular and intracellular concentrations, R=gas constant, T=absolute temperature, P.sub.CPA =cryoprotectant permeability coefficient of the animal cell, .sigma.=the reflection coefficient of said cryoprotectant, wherein V.sub.b =osmotically-inactive cell volume, V.sub.CPA =partial mole volume of said cryoprotectant, 0=initial condition; and determining the size of said cellular volume relative to said predetermined cellular volume limit. ### Claim 20 (depends on 19) The method of claim 19, wherein said cells are sperm cells. ### Claim 21 (depends on 19) The method of claim 19, wherein said cells are blood cells. ### Claim 22 (depends on 19) The method of claim 19, wherein said cells are pancreatic islet cells. ### Claim 23 (depends on 19) The method of claim 19, wherein said cryoprotectant is selected from the group consisting of dimethyl sulfoxide, glycerol, and ethylene glycol. ### Claim 24 A method to remove cryoprotectant from the cells of an animal species, comprising: contacting said cells containing cryoprotectant with a first solution of cryoprotectant and continually lowering the concentration of said cryoprotectant in said first solution at a predetermined rate, said predetermined rate substantially preventing the volumetric excursion of said cells beyond a predetermined upper cellular volume limit, and said predetermined rate being predetermined by determining the cellular volume of said animal cells for a given concentration of said cryoprotectant with differential equations: ##EQU6## wherein J.sub.v =total volume flux, V=cell volume, t=time, N=mole number of the solute, A.sub.c =cell surface area, L.sub.p =water permeability coefficient, C=concentration of solute, J.sub.CPA =cryoprotectant flux across the cell membrane, superscript e=extracellular, superscript i=intracellular, C.sub.CPA =average cryoprotectant concentration of extracellular and intracellular concentrations, R=gas constant, T=absolute temperature, P.sub.CPA =cryoprotectant permeability coefficient of the animal cell, .sigma.=the reflection coefficient of said cryoprotectant, wherein V.sub.b =osmotically-inactive cell volume, V.sub.CPA =partial mole volume of said cryoprotectant, 0=initial condition; and determining the size of said cellular volume relative to said predetermined cellular volume limit. ### Claim 25 (depends on 24) The method of claim 24, wherein said cells are sperm cells. ### Claim 26 (depends on 24) The method of claim 24, wherein said cells are blood cells. ### Claim 27 (depends on 24) The method of claim 24, wherein said cells are pancreatic islet cells. ### Claim 28 (depends on 24) The method of claim 24, wherein said cryoprotectant is selected from the group consisting of dimethyl sulfoxide, glycerol, and ethylene glycol.