a description of an improved system and method for preparing a cryopreserved pool of cells ( e . g . hepatocytes ) from previously cryopreserved hepatocytes obtained from individual donors is described herein . this system and method reduces physical and chemical stress to the hepatocytes during the pooling process while increasing the recovery of viable pooled cells during a post - thaw centrifugation step performed by the end - user . in general , this system and method involves thawing vials of individual donor hepatocytes and pooling them into a preservation solution . the pooled cells are then briefly centrifuged , to pellet both viable and non - viable cells , and then cryopreserved at a high density in multiple vials . the end - user can then perform density gradient centrifugation to separate viable from non - viable cells immediately prior to experimental use . some of the advantages of the proposed method are decreasing the exposure to mechanical , chemical and other environmental factors that reduce the number of viable cells prior to experimenting on the pooled hepatocytes . in one embodiment , previously isolated and cryopreserved hepatocytes from individual donors are stored in liquid nitrogen vapor phase at a minimum of − 150 ° c . individual donor vials to be pooled can be selected at random or based on specific metabolic activity ( e . g . ecod , cytochrome p450 , general phase i or phase ii ), age , race , sex , ethnicity , or other phenotypic determinants . the number of vials thawed depends on the number of individuals included in the mixed population and the size of the pool to be generated . for example , the number of individual vials used for each pool of hepatocytes can be from 2 - 50 individuals . each pool can range from 300 to 1000 vials . for example , a 10 donor pool with 300 vials would require approximately 30 vials from each donor . the individual donor hepatocytes are thawed by submerging the vial in a water bath maintained at 37 ° c . for approximately 2 minutes or until a spindle of ice is barely visible and then the contents are rapidly decanted into a vessel containing a preservation solution at 4 ° c . the preservation solution is used to dilute the dmso ( and / or any other reagents toxic to the hepatocytes ) found in the cryopreservative . in some instances , the preservation solution also provides essential nutrients to the hepatocytes to promote viability during the pooling process . the volume of the preservation solution is dependent on the size of the pool to be generated but can be composed of , for example , a 1 : 1 ratio of preservation solution to 1 ml of cryopreserved cells . the preservation solution can be composed of university of wisconsin solution ( 10 mm potassium lactobionate , 25 mm kh2po4 , 5 mm mgso4 , 30 mm raffinose , 5 mm adenosine , 3 mm glutathione , 1 mm allopurinol , and 50 g / l hyroxyethyl starch ), hypothermosol - base ( hts - base ), hypothermosol - frs ( hts - frs ), or other such medium with or without the addition of fetal bovine serum ( fbs ). the hepatocytes are maintained in the preservation solution at 4 ° c . while multiple vials of hepatocytes are thawed and mixed in the same vessel to create a “ pooled ” population of multiple donor cells . thawed hepatocytes can remain in preservation solution from 2 - 10 hours depending on the number of vials being thawed . after multiple vials of hepatocytes have been thawed and pooled , the cells are centrifuged in a range of 50 - 200 relative centrifugal force ( rcf ) for 8 - 10 min to pellet both viable and non - viable cells . the preservation solution is removed from the pelleted cells for example by aspiration . using a centrifugation step without a density gradient at this point in the process , reduces the physical and chemical stress on the hepatocytes before the second cryopreservation . fig1 a - b illustrate the affect that density gradient ( e . g . percoll ) centrifugation can have on hepatocytes compared to centrifugation without a density gradient . with each subsequent spin , the total number of viable cells was reduced with the density gradient ( percoll ) relative to hepatocytes centrifuged without percoll . these results indicate that centrifugation with a density gradient solution reduces the total number of viable cells . after the cells are pelleted and the preservation solution removed , they are immediately resuspended in a cryprotectant solution which can , for example , be composed of cryostor ® cs10 containing dmso at 10 %. the pooled non - viable and viable hepatocytes are then distributed at 10 - 15 million cells / ml in 1 - 1 . 5 ml aliquots per vial . it should be noted that cell counting can be performed without or with for example trypan blue , acridine orange , or propidium iodide along any of the steps in the process . the combining or resuspending process can be comprised of adding a cryopreservative to the pelleted hepatocytes . the combined pellets and cryopreservative can then be pipetted up and down , vortexed , rocked in a vial back and forth , tapped in a vial or some other similar process to break up the pellets and disperse the hepatocytes throughout the cryopreservative . vials containing pooled hepatocytes are frozen using a controlled rate freezer and maintained in liquid nitrogen at a minimum of − 150 ° c . for at least 3 days and no longer than 10 years prior to shipping . the vials can be shipped on dry - ice or vapor phase liquid nitrogen ( e . g . dewar ) to the end - user and stored in liquid nitrogen at a minimum of − 150 ° c . immediately prior to use , the end - user can thaw the pooled hepatocytes by submerging the vial in a water bath maintained at 37 ° c . for approximately 2 minutes or until a spindle of ice is barely visible . the pooled non - viable and viable hepatocytes cells can be applied to a 20 - 30 % colloidal silica coated with polyvinylpyrrolidone ( percoll ) gradient , and centrifuged through at 50 - 200 rcf for 8 - 10 min to separate viable from non - viable cells . the maximum number of viable cells are therefore isolated immediately prior to use without further exposure to cryopreservative or another freeze - thaw cycle . this process allows for 5 million and up to 8 + million viable cells that can be recovered and immediately used for experimentation . experimentation may include but not be limited to assays for viability ; metabolic activity ; transporter activity ; and xenobiotic uptake , metabolism , efficacy , and toxicity . procedure for the preparation of pooled hepatocytes from individual cryopreserved donor hepatocytes are shown in fig2 and set forth in the following operation : 1 - a . vials of cryopreserved hepatocytes from 10 individual donors are thawed for approximately 2 minutes in a 37 ° c . water bath until a spindle of ice is barely visible . it should be noted , some pools can be composed of between 500 and 900 vials , or in this example 50 to 90 vials per donor . 1 - b . the thawed hepatocyte suspension ( 1 ml ) is pipetted into a 1 l beaker containing 500 ml of hypothermosol - frs preservation solution and maintained at 4 ° c . to generate a hepatocyte pool . 1 - c . the pooled hepatocytes are centrifuged at 100 g for 10 minutes out of the preservative solution ( frs ) to pellet both viable and non - viable cells . 1 - d . the preservation solution is removed by aspiration and the cells are gently resuspended ( e . g . rocked back and forth ) in the cryoprotectant cryostor ® cs10 medium . cells are counted using trypan blue exclusion to determine cells density and additional cryoprotectant solution is added , if needed , to achieve approximately 13 . 3 × 10 ̂ 6 cells per ml . 1 - e . 1 . 5 milliliters or approximately 20 million cells are aliquoted into individual vials . 1 - f . the vials of pooled hepatocytes are cryopreserved in a controlled rate freezer and stored in liquid nitrogen vapor phase at a minimum of − 150 ° c . procedure for the separation of viable cells from pooled hepatocytes as performed by the end - user are show in fig2 and set forth in the following operation : 2 - a . a vial of pooled hepatocytes is thawed for approximately 2 minutes in a 37 ° c . water bath until a spindle of ice is barely visible . 2 - b . the hepatocyte suspension is carefully applied to a 20 - 30 % percoll density gradient 2 - c . the samples are centrifuged at 200 rcf for 10 min to separate viable and non - viable cells 2 - d . a minimum of 5 million viable cells are recovered and used immediately for experimentation it should be noted that yield of viable cells can vary with dilution ratios , and exposure of time during the pooling process . for example , in larger batches it is sometimes cost prohibitive or difficult ( volume of pooling container ) to have a 1 : 1 ratio of preservation solution to thawed cryopreservative . in addition , more time is required to thaw a large number of vials . thus the cells are exposed to increased concentrations of cryopreservative toxins for a longer period of time . it is sometimes easier to do smaller batches , in which the cryopreservative is more dilute and pooling time is reduced , which generally results in higher yields of viable hepatocytes . for example , step 2 - d might yield 5 million viable cells for a larger batch , but up to and greater than 8 million viable cells in a smaller batch . in smaller batches the ratio may be 4 : 1 preservation solution to thawed cryopreservative . the processes herein can be applied to hepatocytes used in suspension or plated . though the examples describe hepatocytes the method and processes could be applied to other cell types . these embodiments and features illustrated and described herein are exemplary but not intended to be limiting nor are the claims listed at the end of this application . multiple combinations and equivalent component parts , ranges and steps are considered within the scope of this application .
the present invention relates to a novel method for the preparation of a pooled or mixed population of cryopreserved cells . in particular , the invention entails the rapid thaw of cells , donated from a single individual , which are mixed to create a heterogeneous population and then cryopreserved . the invention also concerns preparations of multi - cryopreserved cells to increase viability prior to immediate use . the process entails reducing exposure to chemical and physical stresses to increase the resultant number of viable cells .