Systems and methods for the digestion of adipose tissue samples obtained from a client for cryopreservation

Abstract

The present invention is directed to systems and methods of processing aspirated adipose tissue for the isolation of stromal vascular fraction derived stem cells.

1 . A method of isolation of stromal vascular derived stem cells comprising the steps of:
 a. supplying a shipment package comprising a defined client sample container having a first filter and a second filter;   b. inspecting the shipment package components for (i) integrity of the client sample container containing an adipose tissue sample and collection medium and (ii) completed recording information, contained therein;   c. introducing the shipment package components to a processing module of a database via a log-in port by scanning a barcode on the client sample container in the completed recording information;   d. testing the sterility of the adipose tissue sample from the client sample container;   e. removing the remaining collection medium in the client sample container;   f. washing the adipose tissue sample;   g. preparing a digestion solution by diluting an enzyme stock solution based on the amount of enzyme activity;   h. injecting the digestion solution into the sample container containing the adipose tissue sample to form a digestion mixture within the client sample container;   i. incubating the digestion mixture;   j. centrifuging the incubated digestion mixture;   k. withdrawing a stromal vascular fraction phase of the centrifuged digestion mixture, wherein the stromal vascular fraction consists of the fluid phase below the floating mature adipocytes and oil from lysed adipocytes;   l. centrifuging a suspension of the digestion mixture in order to isolate a first stromal vascular pellet from the digestion solution;   m. removing supernatant of the centrifuged suspension isolated in l;   n. re-suspending the first stromal vascular pellet by trituration in red blood cell lysis buffer forming a cell suspension;   o. centrifuging the cell suspension to form a second pellet;   p. removing supernatant of the centrifuged solution isolated in o;   q. re-suspending the second pellet by trituration adding salt solution forming a second suspension;   r. centrifuging the second cell suspension to form a third pellet—comprising a mixture of cells of pre-adipocytes, adipose-derived mesenchymal stem cells, microvascular endothelial cells, endothelial progenitor cells, and monocytes, wherein the mixture of cells either i) does not contain mature adipocytes, and demonstrates a combined viability by acridine organge/propidium iodide or trypan blue dye-exclusion assay of no less than 35%, or ii) at least 1% of the nucleated cells in the mixture are adipose-derived mesenchymal stem cells;   s. retaining the supernatant from the third pellet for a secondary sterility test sample; and   t. proliferating the adipose-derived mesenchymal stem cells of the third pellet in a suitable culture medium under appropriate environmental conditions.   
     
     
         2 . The method of  claim 1 , wherein the sample container comprises three female ports disposed on a top portion of the sample container and one three-way port disposed at a bottom of a tapered section of the sample container, the three-way port including a spike port positioned perpendicular to a bottom side of the sample container, and where each of the ports comprises a removable cap,
 wherein the first filter is position between the top portion and the tapered bottom of the sample container and the second filter is in the center of the tapered bottom proximal to the port located in the center of the tapered bottom,   wherein the method further comprises filtering the digestion mixture via the first filter to allow individual cells of the stromal vascular fraction phase to pass through but excluding large mature adipocytes and smaller cells held together by cellular matrix, wherein the filtering of the digestion mixture initiates at step h. and terminates at step j.   
     
     
         3 . The method of  claim 2 , wherein inspecting the shipment package comprises: ensuring that (a) the sample container is not past a defined expiration date, (b) the adipose tissue sample was collected within the past 36 hours, and (c) the recording information is accurate. 
     
     
         4 . The method of  claim 3 , wherein the method further comprises testing the sterility of the adipose tissue sample after step b. 
     
     
         5 . The method of  claim 4 , wherein preparing a digestion solution comprises the steps of: combining collagenase, neutral protease, and DNase I with a salt solution pre-warmed to 37° C. 
     
     
         6 . The method of  claim 5 , wherein the method further comprises removing a sample of the re-suspended sample of step m. and calculating the total viable cell yield and recording results of the total viable cell yield. 
     
     
         7 . The method of  claim 6 , wherein the method further comprises testing sterility of the supernatant obtained in step p. 
     
     
         8 . The method of  claim 7 , wherein the sample container has at least one port on an upper portion of the sample container and at least one port on a lower portion of the sample container. 
     
     
         9 . The method of  claim 8 , wherein the sample container is made of a material which does not release or leach any potentially toxic substance into stored cells. 
     
     
         10 . The method of  claim 9 , wherein the sample container is made of a material selected from the group consisting of ethyl vinyl acetate, polyethylene, fluoro ethylene propylene and combinations thereof. 
     
     
         11 . The method of  claim 10 , wherein the sample container is sterile and approximately 100 ml to 200 ml in volume. 
     
     
         12 . The adipose-derived mesenchymal stem cells obtained by the method of  claim 1  or  2 . 
     
     
         13 . The adipose-derived mesenchymal stem cells of  claim 12  which comprise phenotypes CD14−, CD19−, CD29+, CD31−, CD34−, CD44+, CD45−, CD49d+, CD73+, CD90+, CD105+, and CD146+, the phenotypes exhibited on 100% of the cells. 
     
     
         14 . The adipose-derived mesenchymal stem cells of  claim 13 , which consistently differentiate into Oil Red+ adipocytes, Alcian Blue+ chondrocytes, and Alizarin Red+ osteocytes.