I want to isolate and culture germ cells of pachytene or pre-pachytene stage from mice testis to study my drug effect, but i can't find any precise protocol to do so. Is it possible to culture germ cells without a feeder layer of somatic cells?
Try on http://worthington-biochem.com/tissuedissociation/Reproductive.html
Study the work of :Hofmann MC, Braydich-Stolle L and Dym M. 2005. Isolation of male germ-line stem cells; influence of GDNF. Dev Biol 279(1): 114-124. PMID: 15708562
She uses the Sta-Put method which filters the cells through a column and works really well
it is not possible until now to culture the pachytene spermatocyte for a long time, but you can isolate it by STAPUT
STAPUT is time consuming and resource intensive. For my work I use FACS isolation based on DNA content. All leptotene till pachytene stages are 4N thus can be sorted higher efficiency and relatively pure populations. Hope it is useful
In our hands working with human testicular biopsies we don´t need to isolate primary spermatocytes but directly stain single cell nuclear spreads with antibodies against SYCP3, SYCP1 and MLH1, for example. Obviously most of cells are negative, but usually we can visualize beautiful synaptonemal complex stainings in primary spermatocytes.
You can use STAPUT to isolate them, it is easy to isolate them, but last long time
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