Collagen-Coated Cellulose Sponge: Three-Dimensional Matrix for Tissue Culture of Walker Tumor 256

Leighton, Joseph; Justh, Gerald; Esper, Martha; Kronenthal, Richard L.

doi:10.1126/science.155.3767.1259-a

Cited by 64 publications

(12 citation statements)

References 4 publications

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“…Collagen seems to be a good substrate for the culture of many types of normal and tumorous cells and tissues (4,5,. Perhaps the flexibility of the collagen-gel substrate permits cells to assume specific shapes that in turn allow cellular growth and differentiation that would not be achievable on a rigid surface such as plastic (40).…”

Section: Discussionmentioning

confidence: 99%

“…Other workers (for example, refs. [3][4][5][6] have stressed the importance of three-dimensional in vitro growth of tumor cells in representing the in vivo situation. For a cultured tumor to be representative of actual cancer, it is essential that the tumor, as it proliferates in vitro, maintain its tissue organization and structure, its oncogenic properties, its differentiated functions, and any cellular heterogeneity that may have been present in vivo (7).…”

mentioning

confidence: 99%

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In vivo-like growth of human tumors in vitro.

Freeman¹,

Hoffman²

1986

Proc. Natl. Acad. Sci. U.S.A.

167

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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 The specialized collagen gel (A.E.F., P. H. Gumerlock, and S. H. Hinrichs, unpublished data) is a commercial product of Health Design Industries (Rochester, NY) and is manufactured from pigskin.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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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

In vivo-like growth of human tumors in vitro.

Freeman¹,

Hoffman²

1986

Proc. Natl. Acad. Sci. U.S.A.

167

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“…To support the extracellular matrix in 3D culture, scaffold-based culture systems have been fabricated and shown to recreate the complex heterologous 3D structure of tumors in vitro (11)(12)(13)(14)(15). These 3D culture technologies have been utilized for the study of OS (13,(15)(16)(17)(18)(19).…”

Section: Discussionmentioning

confidence: 99%

Three-dimensional alginate spheroid culture system of murine osteosarcoma

Akeda

Nishimura²,

Satonaka³

et al. 2009

Oncol Rep

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Abstract. Osteosarcoma (OS) is the most common primary malignant tumor of the bone and often forms pulmonary metastases, which are the most important prognostic factor. For further elucidation of the mechanism underlying the progression and metastasis of human OS, a culture system mimicking the microenvironment of the tumor in vivo is needed. We report a novel three-dimensional (3D) alginate spheroid culture system of murine osteosarcoma. Two different metastatic clones, the parental Dunn and its derivative line LM8, which has a higher metastatic potential to the lungs, were encapsulated in alginate beads to develop the 3D culture system. The beads containing murine OS cells were also transplanted into mice to determine their metastatic potential in vivo. In this culture system, murine OS cells encapsulated in alginate beads were able to grow in a 3D structure with cells detaching from the alginate environment. The number of detaching cells was higher in the LM8 cell line than the Dunn cell line. In the in vivo alginate bead transplantation model, the rate of pulmonary metastasis was higher with LM8 cells compared with that of Dunn cells. The cell characteristics and kinetics in this culture system closely reflect the original malignant potential of the cells in vivo.

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“…It consists of a 1% suspension in fibrillary form (12). A number of commercial sources of collagen are now available, and manuals on methods describe well the preparation of collagen solutions in the individual laboratory.…”

Section: Collagen-coated Cellulose Spongementioning

confidence: 99%

Epithelial tissue formation in matrix culture and in histophysiologic gradient culture

Leighton¹

1992

Journal of Tissue Culture Methods

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SUMMARY: Formation of epithelial tissues in culture so that they become facsimiles in their structure of such tissues in nature requires procedures that comply with several spatial imperatives: a) three-dimensional growth; b) histophysiologic conditions that provide, concurrently, gradients of maturation and of diffusion of metabolites; and c) growth as layers of cells without free edges. Many steps have been required in the evolution of these methods. Two systems are described here in sufficient detail to serve as a manual. Three-dimensional growth of masses of epithelial tissue is accomplished in matrix culture using Gelfoam sponge and collagen-coated cellulose sponge. Radial gradient culture, a recent development, provides conditions that comply with the requirements of histophysiologic gradients and of epithelial tissue growth in layers without interruption in their continuity.

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Collagen-Coated Cellulose Sponge: Three-Dimensional Matrix for Tissue Culture of Walker Tumor 256

Cited by 64 publications

References 4 publications

In vivo-like growth of human tumors in vitro.

In vivo-like growth of human tumors in vitro.

Three-dimensional alginate spheroid culture system of murine osteosarcoma

Epithelial tissue formation in matrix culture and in histophysiologic gradient culture

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