Attachment and culture of dissociated cells from rat embryo cerebral hemispheres on polylysine-coated surface.

Abstract

INTRODUCTION hemispheres from 16 to 18-day old rat embryo were treated with a solution of 0 .125% (wt/vol) trypsin for 5 min, and the resulting cell suspension was aspirated through a Pasteur pipette to complete the dissociation . The cells were centrifuged at 400 g for 5 min and the supernate was discarded . The pellet was then resuspended in the growth medium (13), and the suspension was centrifuged for I min at 200 g . The supernate containing the dissociated single cells was transferred to a sterile bottle, final cell concentration was adjusted to 0 .1 0 .3 x 106 cells per I ml medium, and 3 ml of the above suspension was placed into polylysine-coated Petri dishes . After 30 min incubation at 37uC in an atmosphere of 95% air, 5% CO 2 , the medium containing the nonadhered cells was removed and replaced by fresh medium which was then changed every 3-4 days, depending on its acidity . Phase-contrast microscopy was employed for observation of cultures during their growth period . Cultivation of neural tissue in vitro has been employed in the recent years for studying brain metabolism, isolated from influence by the whole organism. The complex intercellular relationship of the heterogeneous cell types present in the neural tissue have led to the use of several experimental approaches such as tissue explants (I 3) or dissociated single cells prepared by either enzymatic or mechanical means (4-6) . The rationale behind the latter approach is based upon the ability of cells to reassociate and to form well organized aggregates which may acquire some of the characteristics of the original neural tissue (7-10) . However, one of the major disadvantages of the dissociation technique is possible selection for cell types. This could be due either to the dissociation treatment itself or to the inability of certain cell types to attach to the surface when monolayer cultures are employed (11, 12) . In a previous publication (13) we reported that reaggregation of cells from rat cerebral hemispheres occurs before the attachment process . A high percentage of cells which fail to aggregate and to adhere to the plastic surface will then be eliminated operationally at the first change of medium . In this report we present a method for circumventing this selection problem by allowing the majority of cells to adhere . This is achieved by the use of a polylysine pretreated surface, and it is based upon previous observations on the electrostatic interaction of the polycation with the negative charges of the cell membrane (14) .