In vivo-like growth of human tumors in vitro.

Abstract

We show that diverse human tumors obtained directly from surgery or biopsy can grow at high frequency in vitro for long periods of time and still maintain many of their in vivo properties. The in vivo properties maintained in vitro include three-dimensional growth; maintenance of tissue organization and structure, including changes associated with oncogenic transformation; retention of differentiated function; tumorigenicity; and the growth of multiple types of cells from a single tumor.It has not been known whether human tumors can grow at high frequency for long periods of time in vitro and still maintain many oftheir critical in vivo properties. Tumor MATERIALS AND METHODSEstablishment of Human Tumor Explants in Culture. The specialized collagen gel (A.E.F., P. H. Gumerlock, and S. H. Hinrichs, unpublished data) is a commercial product of Health Design Industries (Rochester, NY) and is manufactured from pigskin. The material comes as dehydrated squares, which were removed from their sterile packages and placed in 60-mm plastic tissue-culture dishes containing Eagle's minimal essential medium (MEM) with 10% fetal bovine serum (Irvine Scientific), 0.1 mM nonessential amino acids, and the antibiotics gentamicin (100 ,g/ml) and cefotaxime (95 ,ug/ml). The gels were soaked in this medium with at least one fluid change before use.Immediately after surgery or biopsy, tumor sections in MEM with Hanks' salts and 10% fetal bovine serum were brought to the laboratory. Necrotic tissue was cut away, and the remaining healthy tumor tissue was minced with scissors into '4-mm3 bits. Five to ten of these tumor bits were placed on the collagen surface, where they tended to stick or become embedded in loose fiber structure. Medium was added until the upper part of the gel was reached but not covered. The cultures were refed twice a week.Fixation, Embedding, and Staining of Tumor Cells. Cells growing in the collagen gels were fixed in 10% formalin for at least 48 hr. The fixed material was washed for 1 hr in slowly running tap water to rinse out the formalin and then was processed through a series of changes of ethanol (70-100%), with each change lasting 1 hr. The material was then treated with xylene or chloroform and finally embedded in paraffin. After the paraffin hardened, the material was sectioned at 5Am and then dried for 10-15 min on a slide warmer. The next day the slides were stained with Gill's hematoxylin no. 2 and eosin and then mounted.Autoradiography of Tumor Cells. Cells were labeled metabolically, in the above-described medium used for establishment of tumor explants, with [methyl-3H]thymidine (1 ,uCi/ml, Ci/mmol, ICN; 1 Ci = 37 GBq) for 24 hr. After labeling, the medium was removed and the gels were washed three times for 5 min each with nonradioactive medium. Gels were then fixed, dehydrated, embedded in paraffin, and sectioned. The slides, after deparaffinization with xylene, were coated with Kodak NTB-2 emulsion (diluted 1:1 with water) at 40'C and stored in the dark at 40C for 1 week. The slides ...

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