AU2012100720A4 - Immunomagnetic microbeads for enrichment of CD9+ spermatogonial stem cells - Google Patents

Abstract

Abstract The well documented source for adult multipotent stem cells is spermatogonial stem cells (SSCs) of mammalian testis. It is foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. SSCs isolation from mammalian testis is difficult because of their scarcity and the lack of well characterized cell surface markers. Thus, the isolation of SSCs is of great interest for exploration of spermatogonial physiology and therapeutic approaches for fertility preservation. CD9 is a surface marker expressed in mouse and rat male germline stem cells. In this patent, CD9 positive SSCs were successfully isolated from the sheep testis using enzymatic digestion followed by three step purification: Differential plating, Percoll discontinuous density gradient followed by Magnetic activated cell sorting (MACS). Percoll discontinuous density gradient showed significant differences in the percentage of CD9+ SSCs across individual fraction. Magnetic activated cell sorting of CD9+ cells in the magnetic fraction of sheep testes was in the range of 15-19% which is upto threefolds. CD9+ SSCs were further recovered with appreciable efficiency after immunomagnetic isolation by using various bead: cells ratio in which 4.2:1 ratio gave the highest yield of 69.09 A- 105 with 18% of CD9+ SSCs. Magnetic activated cell sorting using anti-CD9 antibodies provides an efficient and fast approach as compared to conventional approaches such as differential plating and percoll discontinuous density gradient for enrichment strategy for spermatogonial stem cells from sheep testes.

Description

1 Description Spermatogonial stem cells from prior fractionated cells were obtained by using CD 9 antibody coated magnetic beads. In this method a cluster of differentiation-9 (CD-9) surface protein was used as marker of spermatogonial stem cell(SSC) in the sheep. The paramagnetic beads were labelled with anti-CD9 antibody For magnetic sorting of SSC, percoll fractionated cell suspension was split into two aliquots. One aliquot was mixed with anti-CD9-labelled magnetic bead in a ratio of 1:6.1, bead per target cell and incubated for 1.2 hrs at 38.50C. Another aliquot was used as control. The sorting was done by passing this cell suspension in the steel wool column under magnetic field. For this, the steel wool column was prior washed in PBS. The non specific binding sites in the steel wool column were blocked by incubating column in 4.9% bovine serum albumin in phosphate buffered saline(PBS) for 61 mins. The column was then flushed with ice-cold PBS containing 1.01% BSA. Loaded cells were applied to the column under magnetic field. The column was then rinsed three times by 550pl of PBS containing 1.01% BSA, under magnetic field. CD9+ cells carry magnetic bead remain in the matrix of the column as long as it is maintained in the magnetic field. The CD9+cells were finally eluted by removing magnetic field and by rinsing column with PBS with 1.01% BSA, the collected fraction was designated as sorted fraction. The entire sorted fraction so obtained after magnetic sorting were analysed in phase contrast microscope under 200x magnification and the number of unsorted, sorted and depleted cells recorded.