Method of cryopreserving cells

Abstract

A non-linear cooling cryopreservation method for improving cryopreservation protocols for cells that involves producing a simulation of cellular responses to a range of cooling parameters; determining optimal cooling parameters required to minimize cryoinjury to the cells using simulation of cellular responses and experimental results; and incorporating optimal parameters into the protocol. The simulation is based on mathematical models of cellular parameters. A non-linear cooling cryopreservation protocol for cryopreserving stem cells is also disclosed that does not require cryoprotectants.

1 . A method for non-linear cooling cryopreservation of cells comprising determining an optimal cooling profile for maximum recovery of the cells and applying the cooling profile to the cells.  
   
   
       2 . The method for non-linear cooling cryopreservation of  claim 1 , wherein any optimal cooling profile is determined in part or in whole using a simulation of cellular responses to cooling parameters.  
   
   
       3 . The non-linear cooling cryopreservation method of  claim 2 , wherein the cooling parameters comprise temperature, duration of temperature exposure, amount of supercooling, cooling rate and the cryoprotectant and concentration thereof.  
   
   
       4 . The non-linear cooling cryopreservation method of  claim 2 , wherein the cellular responses are selected from the group of responses consisting of but not limited to: 
 a) the maximum degree of intracellular supercooling over the course of the cooling protocol as a predictor of cryoinjury due to intracellular freezing,    b) the maximum intracellular potassium chloride concentration as a predictor of cryoinjury due to exposure to the concentrated solutes.    
   
   
       5 . The non-linear cooling cryopreservation method of  claim 4  wherein the simulation of cellular responses is determined in part or in whole from mathematical models comprising: osmotic transport properties, phase diagrams, and compostition and thermodynamic parameters for the intra- and extra-cellular solutions for a particular cell type.  
   
   
       6 . A non-linear cooling cryopreservation protocol according to  claim 1 , wherein the cryopreservation protocol comprises cooling the cells to a first temperature holding for a first period of time, then cooling the cells to a storage temperature for a second period of time.  
   
   
       7 . A non-linear cooling cryopreservation method according to  claim 6 , wherein the cryopreservation protocol is used with cells stored without cryoprotectants.  
   
   
       8 . A non-linear cooling cryopreservation method of  claim 7  wherein the cryopreservation method is used with cells stored with a pentrating cryoprotectant.  
   
   
       9 . A non-linear cooling cryopreservation method of  claim 8  wherein the cryopreservation method is used with cells stored with infusible permeating cryoprotectant.  
   
   
       10 . A non-linear cooling cryopreservation protocol of  claim 7  wherein the cryopreservation method is used with cells stored with a non-pentrating cryoprotectant.  
   
   
       11 . A non-linear cooling cryopreservation method according to  claim 8 , wherein the non-permeating cryoprotectant comprises but is not limited to sugars, starches, serum, proteins, or plasma.  
   
   
       12 . A non-linear cooling cryopreservation method according to  claim 1 , wherein the cells are selected from the group consisting of: stem cells, progenitor cells, red and white blood cells, sperm cells, oocytes, ova, cells for research or transplant purposes, cellular materials derived from tissues and organs, pancreatic islet cells, chondrocytes, cells of neural origin, cells of hepatic origin, cells of ophthalmolic origin, cells of orthopedic origin, cells from connective tissues, and cells of reproductive origin, and cells of cardiac origin.  
   
   
       13 . A non-linear cooling cryopreservation method according to  claim 12 , wherein the stem cells comprise peripheral blood stem cells, human umbilical cord blood stem cells and stem cells derived from tissues and solid organs or other sources, including fetal and or embryonic sources.  
   
   
       14 . A method for optimizing a method for cryopreservation of cells, the method comprising: 
 a) producing a simulation of cellular responses to a range of cooling parameters;    b) using the information derived from the simulation of cellular responses, determining an optimal cooling profile;    c) using the optimal cooling profile into the cryopreservation method; and optionally    d) further optimizing protocol with results from biological experiments using the simulated optimal cellular responses.    
   
   
       15 . A method according to  claim 14 , wherein the cooling parameters comprise cell temperatures, temperature exposure duration periods, cooling rates, amounts of supercooling, and nature and concentration of cryoprotectants.  
   
   
       16 . A method according to aspect  14 , wherein the simulation of cellular responses comprises calculating cellular responses from mathematical models using osmotic transport properties, phase diagrams, and compostition and thermodynamic parameters for the intra- and extra-cellular solutions for a particular cell type.  
   
   
       17 . A non-linear cooling cryopreservation method optimized according to  claim 14 .  
   
   
       18 . A method for optimizing a cryopreservation protocol for cryopreserving cells wherein the cells are cooled to a first hold temperature for a first period of time, then cooled to a second storage temperature at which the cells are stored for a second period of time before the cells are thawed, the method comprising: 
 a) producing a simulation of cellular responses to a range of cooling parameters based on information derived from a simulation of cellular responses to determine an optimal range of first hold temperature, first range of time periods, and amount of intracellular supercooling and/or amount of intracellular solute concentration required to minimize cryoinjury to the cells; and/or    b) incorporating the optimal first hold temperature, optimal first period of time, and optimal amount of intracellular supercooling into the non-linear cooling cryopreservation method.    
   
   
       19 . A method according to  claim 18 , wherein the cooling parameters comprise cell temperatures, temperature exposure duration periods, cooling rates, amount of supercooling, and nature and amounts of cryoprotectants.  
   
   
       20 . A method according to  claim 18 , wherein the simulation of cellular responses comprises calculating cellular responses from mathematical models using osmotic transport properties, phase diagrams, and composition and thermodynamic parameters for the intra- and extra-cellular solutions for a particular cell type.  
   
   
       21 . A method for the cryopreservation of stem cells, comprising: 
 a) cooling the stem cells to a first temperature holding at that temperature for a first period of time; and    b) cooling the stem cells to a second temperature for storing the cells for a second period of time.    
   
   
       22 . The method of  claim 21  wherein the first temperature is between −5° C. and −30° C., and the first time period is between 1 and 30 minutes.  
   
   
       23 . The method of  claim 21  wherein the second temperature is between −60° C. and −200° C., or is below −60° C.  
   
   
       24 . The method of  claim 21 , wherein the first temperature is between −2° C. and the homogenous nucleation temperature of water and the second temperature is below −60° C.  
   
   
       25 . The method of  claim 24 , wherein the second temperature is between −60° C. and −200° C.  
   
   
       26 . The method of  claim 1  further comprising recovery of the cryopreserved cells by thawing the cells to optimize cell viability.  
   
   
       27 . The use of cells cryopreserved using the method of  claim 1  in transplantation, diagnostics, or in vitro fertilization.  
   
   
       28 . A method for optimizing a cryopreservation protocol for cryopreserving cells wherein cells are cooled in such a way as to maintain a constant amount of intracellular supercooling.  
   
   
       29 . The method of  claim 1  wherein the cells are stem cells.  
   
   
       30 . The method of  claim 29  wherein the amount of constant supercooling is 10° C.  
   
   
       31 . The method of  claim 29  wherein the amount of constant supercooling is between 5° C. and 20° C.  
   
   
       32 . The method of  claim 28  wherein the amount of constant supercooling is between 1° C. and the homogenous nucleotide temperature of water.  
   
   
       33 . The method of  claim 28  wherein the amount of constant supercooling is between 1° C. and 40° C.  
   
   
       34 . The method of  claim 28  that does not comprise the use of cryoprotectants.  
   
   
       35 . The method of  claim 28  that does not comprise the use of permeating cryoprotectants.