SPERM SEPARATION BY ELECTROPHORESIS

Abstract

The present invention relates to the separation of sperm. In particular, the present invention relates to the use of polyvinyl alcohol membranes in the electrophoretic separation of sperm.

Claims

1. A method of using at least one physically cross-linked biocompatible polymeric membrane in the separation of sperm by electrophoresis, wherein the membrane comprises poly (vinyl alcohol) (PVA).

2. The method of claim 1, wherein the membrane comprising PVA does not contain polyacrylamide (PAm)

3. The method of claim 1, wherein the membrane comprising PVA is a restriction membrane that allows passage of an electrical field but does not allow passage of sperm.

4. The method of claim 3, wherein the restriction membrane has a molecular weight cut off (MWCO) of less than 15 kDa.

5. The method of claim 4, wherein the restriction membrane is prepared by a casting and annealing method.

6. The method of claim 1, wherein the membrane comprising PVA is a porous size exclusion membrane (separation membrane).

7. The method of claim 6, wherein the separation membrane has a pore size of less than 10 m.

8. The method of claim 6, wherein the separation membrane is prepared by non-solvent induced phase inversion method followed by post annealing.

9. A method for separating sperm from a first solution into a second solution, the method comprising the steps of: providing the first solution to a membrane stack comprising a separation membrane disposed between first and second physically cross-linked biocompatible polymeric restriction membranes comprising PVA, wherein the first solution lies between the first restriction membrane and the separation membrane and wherein the separation membrane has a preselected pore size; and applying an electrical field across the stack, wherein sperm in the first solution that have a negative charge and are smaller than the preselected pore size move towards the anode and pass through the separation membrane into the second solution which lies between the separation membrane and the second restriction membrane.

10. The method of claim 9, wherein the restriction and separation membranes do not contain PAm.

11. The method of claim 9, wherein the separation membrane is a physically cross-linked biocompatible polymeric separation membrane comprising PVA.

12. A method for separating sperm from a first solution into a second solution, the method comprising the steps of: separating the first and second solutions by means of a physically cross-linked biocompatible polymeric separation membrane comprising PVA, wherein the separation membrane has a preselected pore size; and applying an electric field across the first and second solutions, wherein sperm in the first solution that have a negative charge and are smaller than the preselected pore size move towards the anode and pass through the separation membrane into the second solution.

13. The method of claim 12, wherein the separation membrane does not contain PAm.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0080] FIG. 1: Operating principle of electrophoretic separation. Fractionation of macromolecules or cells is achieved either through differences in macromolecular charge or electrophoretic mobility; or membrane size exclusion due to differences in macromolecular radius.

[0081] FIG. 2: Operating principle of electrophoretic sperm separation. Viable sperm migrate towards the positive cathode and pass through a separation membrane.

[0082] FIG. 3: CS10 electrophoresis separation apparatus.

PREFERRED EMBODIMENT OF THE INVENTION

[0083] Although the invention has been described with reference to certain embodiments detailed herein, other embodiments can achieve the same or similar results. Variations and modifications of the invention will be obvious to those skilled in the art and the invention is intended to cover all such modifications and equivalents.

[0084] The present invention relates to the use of PVA membranes in the electrophoretic separation of sperm.

[0085] The use of PVA has several advantages for use in the separation of sperm: PVA is hydrophilic which may reduce membrane fouling by non-specific protein adsorption; it is biocompatible and neutral, aiding fabrication of membranes with low electroendosmosis (flow of bulk fluids through a membrane caused by an applied potential across membrane).

[0086] A membrane-based electrophoresis apparatus typically includes a cartridge which houses a number of membranes forming at least two chambers, cathode and anode in respective electrode chambers connected to a suitable power supply, reservoirs for samples, buffers and electrolytes, pumps for passing samples, buffers and electrolytes, and cooling means to maintain samples, buffers and electrolytes at a required temperature during electrophoresis. The cartridge contains at least three substantially planar membranes disposed and spaced relative to each other to form two chambers through which sample or solvent can be passed. A separation membrane is disposed between two restriction membranes. When the cartridge is installed in the apparatus, the restriction membranes are located adjacent to an electrode. An example of a cartridge is described in AU 738361, Descriptions of membrane-based electrophoresis can be found in U.S. Pat. Nos. 5,039,386 and 5,650,055. An apparatus particularly suitable for use in isoelectric separation applications can be found in WO 02/24314.

[0087] One electrophoresis apparatus suitable for use in the present invention comprises:

[0088] (a) a first electrolyte chamber;

[0089] (b) a second electrolyte chamber,

[0090] (c) a first sample chamber disposed between the first electrolyte chamber and the second electrolyte chamber;

[0091] (d) a second sample chamber disposed adjacent to the first sample chamber disposed and between the first electrolyte chamber and the second electrolyte chamber;

[0092] (e) a separation membrane disposed between the first sample chamber and the second sample chamber, the separation membrane preventing substantial convective mixing of contents of the first and second sample chambers;

[0093] (f) a first restriction membrane disposed between the first electrolyte chamber and the first sample chamber, the first restriction membrane preventing substantial convective mixing of contents of the first electrolyte chamber and the first sample chamber;

[0094] (g) a second restriction membrane disposed between the second sample chamber and the second electrolyte chamber, the second restriction membrane preventing substantial convective mixing of contents of the second electrolyte chamber and the second sample chamber; and

[0095] (h) electrodes disposed in the first and second electrolyte chambers.

[0096] The electrophoresis apparatus may further comprise one or more of:

[0097] (i) an electrolyte reservoir;

[0098] (j) a first sample reservoir and a second sample reservoir;

[0099] (k) means for supplying electrolyte from the electrolyte reservoir to the first and second electrolyte chambers; and

[0100] (l) means for supplying sample or liquid from at least the first sample reservoir to the first sample chamber, or from the second sample reservoir to the second sample chamber.

[0101] The apparatus may further comprise:

[0102] (m) a first electrolyte reservoir and a second electrolyte reservoir; and

[0103] (n) means for supplying electrolyte from the first electrolyte reservoir to the first electrolyte chamber and electrolyte from second electrolyte reservoir to the second electrolyte chamber.

[0104] The apparatus may further comprise one or more of:

[0105] means for circulating electrolyte from the electrolyte reservoir(s) through the electrolyte chambers forming electrolyte streams in the electrolyte chambers; and

[0106] means for circulating contents from each of the first and second sample reservoirs through the respective first and second sample chambers forming first and second sample streams in the respective sample chambers;

[0107] means for removing and replacing sample in the first or second sample reservoirs; and

[0108] means to maintain temperature of electrolyte and sample solutions.

[0109] Preparation and characterisation of PVA restriction and separation membranes is described in detail in U.S. Provisional Application No. 62/326,331 filed on 22 Apr. 2016, the content of which is herein incorporated by reference in its entirety. In brief, PVA restriction membranes were prepared by casting and annealing with the predominant stabilising mechanism in the restriction membranes being non-covalent cross-linking due to the formation of crystalline domains, and PVA separation membranes were prepared by a combination of casting and non-solvent induced phase inversion to generate a porous structure, followed by post annealing.

[0110] The present invention is further described by the following non-limiting examples.

EXAMPLES

Example 1PVA Restriction Membrane Preparation

[0111] To prepare a PVA stock solution, a known quantity of high-purity water was added to a round bottom flask. The flask was placed in a thermostated oil bath (90 C.) and stirred under reflux. A known mass of PVA was added portions, until the desired concentration (% w/w) was achieved. The slurry was stirred at 90 C. until no undissolved gels were observed, forming a viscous solution. Following, the solution was stirred an additional 30 min at reflux to ensure complete dissolution of PVA. The solution was cooled slowly to room temperature with gentle mixing, then weighed. If necessary, high-purity water was then added to return the net mass to the starting mass to produce the desired concentration (% w/w). Following, the solution was gently stirred at room temperature for 30 minutes, and then stood overnight without stirring to remove entrained bubbles.

[0112] Stock solutions of poly(ethylene glycol) (PEG) or poly(N-vinylpyrrolidone) (PVPON) in water were prepared by dissolving appropriate PEG and PVPON in water to the desired concentration (% w/w).

[0113] PVA restriction membranes were prepared by casting and annealing method. The membrane casting solutions were prepared by mixing an appropriate mass of PVA solution, additive solution (PEG or PVPON, varied MW) and high-purity H.sub.2O. The solutions were stirred for a minimum of 30 minutes to ensure all components were well mixed, then stood for 30 minutes at room temperature to allow any entrained bubbles to be collected. The membranes were cast into a membrane casting tank comprising two layers of PET substrates. Following air-drying, the membranes were annealed at 110 C. for 1 hr, then swollen for 48 hours in high-purity H.sub.2O before analysis. The membrane formulations detail is listed in Table 1.

TABLE-US-00001 TABLE 1 Formulations of PVA restriction membranes Annealing at Batch No PVA (% w/w) PEG (% w/w) 110 C. PVA-1 PVA.sub.89k (10%) 0% Yes PVA-4 PVA.sub.22k (15%) 0% Yes PVA-18 PVA.sub.22k (14%) PEG.sub.8k (1%) Yes

Example 2PVA Separation Membrane Preparation

[0114] PVA, PEG and PVPON stock solutions were prepared as described for restriction PVA membranes.

[0115] PVA separation membranes were prepared by non-solvent induced phase inversion. In the first instance, the membranes with varying pore sizes were prepared without any support substrates for ease of characterisation. The membrane casting solutions were prepared by mixing an appropriate mass of PVA solution (89 kg/mol), additive solution (PEG or PVPON, varied MW) and high-purity H.sub.2O. The solutions were stirred for a minimum of 30 minutes to ensure all components were well mixed, then stood for 30 minutes at room temperature to allow any entrained bubbles to be collected. The membranes were cast into machined wells formed in stainless steel discs (internal diameter=55 mm, well depth=1 mm), air-dried for 5 minutes, and then immersed in a non-solvent coagulation bath (200 mL).

[0116] The composition of the non-solvent bath was varied by altering the volume ratios of H.sub.2O, methanol (MeOH) and ethanol (EtOH). The membranes were held in the coagulation bath for a minimum for 60 minutes. To reduce membrane shrinkage and pore collapse upon drying, after coagulation, the membranes were immersed in an acetone bath (100 mL) for 15 minutes, then removed and placed on a glass plates and air-dried. Following air-drying, the membranes were annealed at 110 C. for 1 hr, then swollen for 48 hours in 2100 mL portions of high-purity H.sub.2O before analysis. The PVA separation membrane formulations with PEG additives are shown in Table 2. Strength of hydrated membranes fabricated from high molecular weight PVA (89-98 kg/mol) is very good; can be manipulated easily with tweezers, can be stretched.

TABLE-US-00002 TABLE 2 Formulation table for PVA-64, PVA-66 and PVA-67 separation membranes. PVA Additive Annealing Batch No (% w/w) (% w/w) Phase Inversion at 110 C. PVA-64 PVA.sub.89k PEG.sub.2k 64:21:15 v/v Yes (11.0%) (0.91%) EtOH/MeOH/H.sub.2O, 90 min PVA-66 PVA.sub.89k PEG.sub.2k 21:64:15 v/v Yes (11.0%) (0.91%) EtOH/MeOH/H.sub.2O, 90 min PVA-67 PVA.sub.89k PEG.sub.2k 85:15 v/v Yes (11.0%) (0.91%) MeOH/H.sub.2O, 90 min

[0117] PVA separation membrane formulations with PVPON additive are shown in Table 3.

TABLE-US-00003 TABLE 3 Formulation table for PVA-80, PVA-81 PVA-83 separation membranes PVA Additive Annealing at Batch No (% w/w) (% w/w) Phase Inversion 110 C. PVA-80 PVA.sub.89k PVPON.sub.10k 85:15 v/v Yes (11.8%) (0.91%) MeOH/H.sub.2O, 90 min PVA-81 PVA.sub.89k PVPON.sub.10k 6.5:78.5:15 v/v Yes (11.8%) (0.91%) EtOH/MeOH/H.sub.2O, 90 min PVA-83 PVA.sub.89k PVPON.sub.10k 19.5/65.5:15 v/v Yes (11.8%) (0.91%) MeOH/H.sub.2O, 90 min

Example 3Use of PVA Membranes in the Separation of Sperm

[0118] The NuSep CS10 electrophoresis apparatus was used to carry out tests to compare PAm and PVA restriction membranes. A schematic diagram of the CS10 apparatus is shown in FIG. 3. The CS10 apparatus enables the precise control of run time, voltage and circulating buffer rates and is suitable for the processing of a diverse range of samples.

[0119] Restriction and separation membranes were cut into the required size using an appropriate die and assembled in a cartridge, wherein the cartridge comprises a separation membrane sandwiched between two restriction membranes. The cartridge was inserted into the CS10 apparatus for electrophoresis.

Electrophoresis of Bovine Sperm

[0120] Electrophoresis of bovine sperm samples was performed in the CS10 apparatus with a 8 m polycarbonate separation membrane and either (a) PAm restriction membranes or (b) PVA restriction membranes. Samples were processed at 35 volts, a maximum current of 50 mA and 5 minutes running time. Original, harvested and retained/residual samples were stained with Eosine-Nigrosine (EN) to assess sperm viability and stained with Aniline Blue (AB) to assess DNA fragmentation. Motility was assessed by eye under 100 light microscope.

[0121] The results for PAm restriction membranes are presented in Table 4.

TABLE-US-00004 TABLE 4 Results for bovine sperm separation with PAm restriction membranes Sperm count Harvest EN stain AB stain Motility (10.sup.6 cells/ml) rate (%) Live (%) Negative (%) (%) O H R H R O H R O H R O H R E1 56 9.3 43.3 19 78 77 89 65 97 98 94 64 85 62 E2 43 7.5 36.5 17.3 84.9 37 59 28 99 100 100 26 52 12 E3 12 2.4 10.5 20 87.5 67 81 54 100 100 100 58 75 36 (O = original, H = harvested and R = retained/residual)

[0122] The results for PVA restriction membranes are presented in Table 5.

TABLE-US-00005 TABLE 5 Results for bovine sperm separation with PVA restriction membranes Sperm count Harvest EN stain AB stain Motility (10.sup.6 cells/ml) rate (%) Live (%) Negative (%) (%) O H R H R O H R O H R O H R E1 22 6.6 13.5 30.2 61.4 72 86 74 100 100 100 71 90 63 E2 28 6.8 16.7 24.2 59.6 68 83 71 100 100 100 73 86 56 E3 31 6.7 21.6 21.5 69.7 71 80 66 100 100 100 69 87 50 (O = original, H = harvested and R = retained)

Electrophoresis of Human Sperm

[0123] Electrophoresis of human sperm samples was performed in the CS10 apparatus with a 8 m polycarbonate separation membrane and either (a) PAm restriction membranes or (b) PVA restriction membranes. Samples were processed and analysed as described above.

[0124] The results for PAm restriction membranes are presented in Table 6.

TABLE-US-00006 TABLE 6 Results for bovine sperm separation with PAm restriction membranes Sperm count Harvest EN stain AB stain Motility (10.sup.6 cells/ml) rate (%) Live (%) Negative (%) (%) O H R H R O H R O H R O H R E1 23 4.5 18.4 19.6 79.4 61 83 35 82 95 63 64 86 43 E2 19 3.9 15.4 20 80 72 96 23 84 95 66 67 92 34 E3 52 9.5 23.5 18 46 61 78 43 64 90 32 45 73 32 (O = original, H = harvested and R = retained/residual)

[0125] The results for PVA restriction membranes are presented in Table 7.

TABLE-US-00007 TABLE 7 Results for bovine sperm separation with PAm restriction membranes Sperm count Harvest EN stain AB stain Motility (10.sup.6 cells/ml) rate (%) Live (%) Negative (%) (%) O H R H R O H R O H R O H R E1 21 3.9 16.2 19.5 77.5 59 81 33 61 85 50 62 85 35 E2 20 3.6 15.8 18 79 63 84 40 55 75 35 55 74 33 (O = original, H = harvested and R = retained/residual)

Comparison of PAm and PVA Membranes

[0126] A comparison of PAm and PVA restriction membranes in the separation of bovine and human sperm is presented in Table 8.

TABLE-US-00008 TABLE 8 Comparison of PAm and PVA restriction membranes in the separation of bovine and human sperm Harvest EN stain Motility rate (%) Live (%) (%) Bovine PAm 18.8 16 21 Bovine PVA 25.3 12 16 Human PAm 19.2 21 25 Human PVA 18.75 22 21

[0127] No statistically significant differences were observed between PVA and Pam restriction membranes for the separation of bovine or human sperm. Accordingly, PVA restriction membranes may be substituted for PAm restriction membranes for sperm separation.