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Organotypic culture and Transplantation tools

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Functional analysis of isolated ceIIs and tissues requires growth conditions enabling the expression of specific functions under experimental conditions in vitro. Organotypic cuIture models comprising different extracellular matrices as culture substratum often enriched with mesenchymal ceIIs provide improved environmental conditions for tissue-specific ceII differentiation and function. Having initially been developed for squamous (e.g. epidermis) or transport (e.g. kidney) epithelia, various modifications of these culture systems have been used for maintaining organotypic growth and differentiation of many normal and tumor cells. The Combi Ring Dish {CRD) System consisting of loosely fitting concentric teflon rings is a unique holding device for various extraceIIular matrices ranging from coated filters over collagen gels to biologic membranes (e.g, lens capsule). Although other culture devices have been developed, the CRD is unique in that it is simple, versatile and applicable to provide various tissuespecific extracellular matrices and growth conditions. CRD (Combi Ring Dish) Teflon rings, Silicone Transplan tation Chambers Perforated Polypropylene Film and Stanzen Petri Dish.

In such organotypic cocultures of keratinocytes and fibroblasts embedded in collagen matrix a rather nor alepidermis is reconstructed.

In spite of many improvements made in these in vitro models, structural architecture and complex functions of most normal organs and derived tumors cannot yet be adequately produced in vitro but require in vive environment. By transplantation of intact celllayers preformed in organotypic cultures the development of different epithelial cells into complete well-structured tissues can be achieved and their functions studied in these heterotransplants and these can be used as in vive controls to culture models. The silicone transplantation chamber is an excellent tool for protection of surface transplants against infection, dessication, shrinkage, and replacement by hast skin. Cells and tissues can be transplanted by placing them directly on host tissue (muscle fascia or induced granulation tissue) or by transfer of preformed organotypic cultures attached to their specific matrix when growing in CRD cultures.


Epithelial-Mesenchymal Interactions in Surface Transplants

keratinocytes

HaCaT

benign

malignant

Schematic cross sections on transplants of normal, immortalized and benign or malignant keraticocytes. Development of a normal epidermis 2 week after transplantation of primary skin keratinocytes on nude mice.

Moerover, early stages of tumor growth and typical host interactions such as mesenchyme activation, angiogenesis and invasion can be investigated in such transplants under defined in vivo conditions. This is a rapid and sensitive tumorigenicity assay discriminating benign from malignant (invasive) cells. (see Fig.5)

Fig.5 Stages of tumor stroma interactions seven days malignant human keratinocytes on nude mice a) H a.E.staining; b) immunostaining of blood vessels (red) and nucIei (blue)

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From the full-thickness human salpinx wall or, altematively, frorn the endometrium a tissue disk was punched out. The tissue fragment was graf ted onto the exposed full-skin defect of a severe combined immunodeficient (SCID) mouse with the mucosal epithelium facing outside. The .Iiat-Iike" upper part of a silicone transplantation chamber was pulled over the transplant and pressed onto the fascia. The brim of the chamber was placed undemeath the adjacent mouse skin and the wound margins were fixed with two suture cIamps. Phosphatebuffered salinewas injected into the silicone chamber to provide a humid milieu for the mucous membrane surface. The female genital mucosa as weil as the underlying stromal tissue with capillaries of human origin could be preserved for many weeks in this SCID mouse model. This system allows the study of the interactions of test substances, or of invasive, pathogenic microorganisms, with human epithelial cells under in vivo conditions.(FigA.)

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Pelri dl~n-

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Extracellular matrix in combination with microporous filter membranes can be used as an in vitro model system to study the mechanisms goveming cell migration through basallaminae for malignant cells. Polycarbonate filter membranes mounted in combi-ring dishes were coated with extra-cellular matrix (ECM) on the underside. The filter chambers were filled with tumour cell suspension. (Fig.l) -Erdei M, Spiess E, Trefz G, Boxberger HJ, and Ebert W (1 992), Anticancer Research 12: 349-360. Microporous filter membranes mounted in combi-ring dishes can be used in order to study the extravasation of tumour cells through an endothelial cell layer. The endothelial cells were seeded on the underside of the filter and grown to confluency. The tumour cells were placed into the filter chambers. (Fig.2) As an alternative to the culture on artificial substrata, natural bovine Iens capsules were mounted in combi-ring dishes to create basement membrane chambers. While on artificial supports endothelial and epithelial cells were only minimally polarized, a higher degree of polarity was observed for the cells cultured in lens capsule chambers. (Fig.3) -Boxberger HJ, Paweletz N, Spiess E, and Kriehuber R (1989), Anticancer Research 9: 1777-1786, -Boxberger HJ and Paweletz N (1990), Anticancer Research 10: 741-752. -Boxborger HJ and Paweletz N (1990), Anticancer Research 10: 1265-1274. -Boxberger HJ, Sessler MJ, Maetzel B, and MeyerTF (1993), Eur. J. Gell Blot. 62:140-151. -Boxberger HJ, Sessler MJ, Maetzel B, Mosleh IM, and MeyerTF (1994), Epith, Cell Biol. 3: 85-95. -Boxberger HJ and Meyer TF (1995) Biot.Ce1l82: 109-119. Pieces of salpinx wall or endometrium were graf ted onto skin defects of SCID mice. Transplantation chambers were pulled over the transplants which survived for more than one month after surgery and retained their typical epithelial surfaces. (FigA) -Kaufmann R, Rudolphi A, Boxberger Hj, Hainzl A, Rosenthai H, and Reimann J (1 995), Gynecol. Obstet. lnvest. 40: 97-100.

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In addition, pharmacologic responses of normal and transformed human cells growing under in vivo conditions can be studied in this assay following topical and/or systemic application of agents.

30900

FK U

upper part

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6

16

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30901

FKL

lower part

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5,8

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30911

FGU

upper part

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8

18

9,9

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30912

FGL

lower part

0,38

7

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5,5

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30266

F1 U

upper part

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4

13

7,0

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30267

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lower part

0,07

3

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lower part

0,94

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F3 U

upper part

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lower part

8,04

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30272

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Stanzen petridish

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35

10,0

20

30273

F5

glass dish

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13 24 42

3,0 3,0 3,0

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*) size used in references

30902

Typ A

Combi-Ring Dish (CRD)

8

9,5

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30903

TypB

Combi-Ring Dish (CRD)

10

11,5

10

30904

Typ

C

Combi-Ring Dish (CRD)

12

13,5

10

30905

TypD

Combi-Ring Dish (CRD)

14

15,5

10

30906

Typ

a

Combi-Ring Dish (CRD)

6,5

8

10

30907

Typ b

Combi-Ring Dish (CRD)

8,5

10

10

30908

Typ c

Combi-Ring Dish (CRD)

10,5

12

10

30909

Typ d

Combi-Ring Dish (CRD)

12,5

14

10

30910

Typ

Combi-Ring Dish (CRD)

10,5

12

10

c

,

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Chambers  

Silicone culture and transplantation chambers

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