Sperm processing method, apparatus and related media compositions

Abstract

Embodiments of the present invention relate generally to processes, systems, and compositions useful in manipulating a ratio of viable X chromosome bearing sperm to viable Y chromosome bearing sperm in at least one sperm population and useful for preserving the resulting manipulated sperm population. In some embodiments a cryoprotectant may be incorporated into various medias used in manipulating the sperm sample, such as in a staining media, a sheath fluid, and a collection media.

We claim: 
     
         1 . A method of freezing a manipulated sperm sample comprising:
 contacting a stained sperm sample with a sheath fluid in a flow path;   manipulating a ratio of viable X-chromosome bearing sperm to viable Y-chromosome bearing sperm to form at least one manipulated sperm population;   collecting the manipulated sperm sample in the presence of a polyalcohol;   concentrating the collected sperm sample;   resuspending the concentrated sperm sample in a freezing extender; and   freezing the resuspended sperm sample.   
     
     
         2 . The method of  claim 1 , further comprising the step of cooling sperm for a period of 120 minutes or a period of less than 120 minutes. 
     
     
         3 . The method of  claim 1 , wherein the step of collecting a mixture further comprises: collecting the mixture in a container. 
     
     
         4 . The method of  claim 3 , wherein the step of collecting a mixture further comprises: collecting the mixture in a container having between about 5 ml and about 50 ml of collection media. 
     
     
         5 . The method of  claim 3 , wherein the step of collecting a mixture further comprises collecting a sorted sperm sample from a flow cytometer until the container is filled to between about 60% to about 90% of the container's capacity. 
     
     
         6 . The method of  claim 5 , wherein the step of concentrating the collected mixture is performed in the container. 
     
     
         7 . The method of  claim 1 , wherein the step of concentrating the collected sperm sample follows the step of collecting the manipulated sperm sample in the presence of a polyalcohol without any further dilution occurring between the steps. 
     
     
         8 . The method of  claim 1 , wherein the freezing extender comprises less than 6% polyalcohol vol./vol. or wt./vol. 
     
     
         9 . The method of  claim 1 , wherein the freezing extender comprises between 3.5% and 5.5% polyalcohol vol./vol. or wt./vol. 
     
     
         10 . The method of  claim 1 , wherein the freezing extender comprises between 4% and 5% polyalcohol vol./vol. or wt./vol. 
     
     
         11 . The method of  claim 1 , wherein the resuspended mixture is held for between 2 hours and 18 hours before freezing. 
     
     
         12 . The method of  claim 1 , wherein the step of collecting the manipulated sperm sample in the presence of a polyalcohol further comprises adding the polyalcohol to the sperm sample during a step of staining. 
     
     
         13 . The method of  claim 1 , wherein the step of collecting the manipulated sperm sample in the presence of a polyalcohol further comprises adding the polyalcohol to the sheath fluid which is collected together with the sperm sample in the step of collecting. 
     
     
         14 . The method of  claim 1 , wherein the step of collecting the manipulated sperm sample in the presence of a polyalcohol further comprises collecting the manipulated sperm sample in a container having a collection media, the collection media comprising the polyalcohol. 
     
     
         15 . The method of  claim 1 , wherein the step of collecting the manipulated sperm sample in the presence of a polyalcohol further comprises incorporating the polyalcohol into one or more of: a staining media used to stain the sperm sample, the sheath fluid, and the collection media. 
     
     
         16 . The method of  claim 1 , wherein the step of manipulating a ratio of viable X-chromosome bearing sperm to viable Y-chromosome bearing sperm to form at least one manipulated sperm population further comprises classifying cells in the flow path and selecting sperm based on their classification. 
     
     
         17 . The method of  claim 1 , wherein the step of manipulating a ratio of viable X-chromosome bearing sperm to viable Y-chromosome bearing sperm to form at least one manipulated sperm population further comprises either forming droplets expected to contain selected sperm and deflecting those droplets for collection or photo-damaging selected sperm in the flow path.