Neuroscience Letters, 48 (1984) 191-196 Elsevier Scientific Publishers Ireland Ltd. NSL 02800 REAGGREGATION OF EMBRYONIC CHICK RETINA CELLS: PIGMENT EPITHELIAL CELLS INDUCE A HIGH ORDER OF STRATIFICATION 191 G. VOLLMER, P.G. LAYER and A. GIERER Max-Planck-Institut fiir Entwicklungsbiologie, Spemannstr. 35, D-7400 Tiibingen (F.R.G.) (Received March 6th, 1984; Revised version received and accepted April 1lth, 1984) Key words: chick retina - cell aggregates - sorting out - lamination - retinoids - pigment epithelium We report here that, in comparison to aggregates from retinal cells alone, addition of pigmented epithelial cells to retinal cells in rotary culture results in a pronounced increase of spatial order. A par- ticularly high level of organization is found in about 15-20°70 of the aggregates. In these 'retinoids' the main layers characteristic of developing in vivo retinae can be distinguished in correct sequential arrange- ment on the basis of morphological criteria and by using acetylcholinesterase histochemistry [5, 6, 15], peanut agglutinin-lectin binding [11] and Lucifer Yellow staining [7-9]. Dissociated single cells from embryonic chick retinae reaggregate quickly and form relatively organized histotypic structures in a rotary culture system. Reag- gregation of dissociated single cells starts with a random alignment of retinal cells, followed by the formation of primary rosettes and by the incorporation of addi- tional cells into the aggregate [1, 4, 10-14]. We studied the internal order of these aggregates upon prolonged incubation of 2-3 weeks. Fujisawa [3] described specific layers which are formed upon aggregation of retinal cells on the chorioallantoic membrane. We were able to demonstrate a similar high degree of laminar organiza- tion in a pure in vitro system of aggregates derived from mixed cultures of retinal and pigmentepithelial (PE) cells. Methodologically, the neuroretinae and pigmented epithelia from E6 chicken em- bryos (White Leghorn) were dissected and washed with Hank's solution. Pigmented epithelia were first incubated in 1 mg/ml collagenase and 300 U/ml hyaluronidase (both from Boehringer) in Eagle's Minimal Essential Medium (Ea-MEM) for 10 min at 37°C, washed once in Hank's solution and treated with 1 mg/ml trypsin (Difco) for 20 min at 37°C. Isolated retinae were digested only by trypsin (10 min, 20°C and 10 min, 37°C). Then the tissues were rinsed in Ea-MEM and mildly dissociated into single cells in the presence of 0.05 mg/ml DNAase. After a 3-fold wash in Ea- MEM the cells were resuspended in aggregation medium (10070 fetal calf serum, 2°70 chicken serum, 1 070 glutamine, 0.1 070 penicillin/streptomycin and 0.02 mg/ml gen- tamycin in Ea-MEM). A 2 ml suspension of cells consisting either of 2-5 x 106 retina 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.