DE202016000904U1 - Cell line of horse tissue and its application - Google Patents

Abstract

Cell line of horses tissue, characterized in that it comprises epithelial cells from the bronchi and is available under conventional adhesion culture and in the air-liquid interface.

Description

The present invention relates to a cell line of primary epithelial cells isolated from the bronchial mucosa of horses. The aim of the invention is to cultivate primary equine bronchial epithelial cells which are used in various in vitro assays, inter alia, for the detection of the efficacy of substances on the epithelial barrier and the differentiation and function of the respiratory epithelium of horses.

In respiratory diseases a damaged respiratory epithelium is frequently to be observed, which is characterized by an increased exfoliation of the cells, a reduction of the ciliary cells and an increase of slime-forming goblet cells as well as a reduced barrier function by loss of cell-cell-contacts (tight-junctions). Especially in the Recurrent Airway Obstruction (RAO) of the horse such changes are regularly observed. They have a lasting effect on the course of the disease because the damaged barrier can cause noxae and pathogens to enter the respiratory tract and cause inflammation and hyper-responsiveness of the bronchi. So far, this process known as airway remodeling in the equine respiratory tract is being researched exclusively in vivo on experimental animals. However, such studies are narrow limits for ethical and cost reasons.

In human medicine, various cell culture models are available that can be used to investigate which processes in the respiratory tract of humans lead to the loss of ciliary cells and the metaplasia of goblet cells and to the reduction of tight junctions. Here, the adherent culture on collagen-coated culture plates / bottles is the most widely used culture method in which studies on growth and differentiation of the cells can be performed. In addition, primary, human bronchial epithelial cells are also cultured in the so-called air-liquid interface (ALI) on semipermeable membranes ( DE 10 2004 024 834 A1 ). In addition to the applications already mentioned, this culture method also makes it possible to measure the epithelial barrier function ( CA 2508256 ). The culture of epithelial cells in ALI is characterized in that the cells are cultured on a collagen-coated, semipermeable membrane (insert), wherein the insert is suspended in a well of a culture plate. In the basolateral compartment (outside the insert) is the medium, so that the cells of basal (below) are supplied with nutrients, while they have contact on the apical side (top) to the gas phase. In this way, a model is created that reproduces the in-vivo situation in the respiratory tract very well and supports the differentiation of cultured epithelial cells into beaker and ciliated cells. Studies on the pathogenesis of respiratory diseases in humans as well as on the effect of drugs and the toxicological effects of substances are already successfully carried out on these models. Human and other species epithelial cell lines, such as mouse ( CA 2445961 A1 ) or rat ( CN 102787095 ) can only partially depict the functional features of the respiratory epithelium of horses. Although there are already approaches to the culture of equine respiratory epithelial cells, but so far there is no suitable bronchial epithelial cell line from the horse.

It is therefore an object of the present invention to obtain and cultivate primary equine bronchial epithelial cells (EBEC) that differentiate them into bronchial carcinoma phenotypes of horses and form an intact tight junction epithelial barrier to provide a functional, pharmacological and functional test system To establish toxicological studies under conventional and air-liquid culture according to claim 1 and 2.

The object is achieved according to claims 3-5 by the enzymatic isolation of epithelial cells from the bronchi of horses and the culture of the isolated cells on collagen-coated culture plates and semipermeable membranes (ThinCert ^® Greiner BioOne) and by in vitro assays for the study of drugs the proliferation and barrier function of epithelial cells according to claims 7-10 dissolved.

• 1. Behandlung der Epithelzellen mit Arzneimitteln/Schadstoffen/Entzündungsmediatoren unmittelbar an der basolateralen und/oder apikalen Seite.
• 2. Nachweis der Wirksamkeit von Arzneimitteln/Schadstoffen/Entzündungsmediatoren auf die epitheliale Barriere
• 3. Untersuchung der wachstumsstimulierenden und wachstumsinhibierenden Wirkung von Wirkstoffen.
• 4. Analyse von freigesetzten Zellausscheidungsprodukten während entsprechenden Experimenten.
• 5. Zelluläre Interaktionen durch Cokultivierung verschiedener Zelltypen, z. B. mit Fibroblasten oder Neutrophilen Granulozyten.
• 6. Anheftungsverhalten der Epithelzellen auf veränderter Kollagenmatrix, wobei die Zusammensetzung analog zur Extrazellularmatrix in entzündlich veränderten Atemwegen angepasst werden kann.

The advantage of this EBEC cell line according to the invention is that the epithelial cells originate from the respiratory tract of horses, can be stably kept in culture for a long period of time and for the first time enable the functional investigation of airway remodeling in the in-vitro system from the horse. Various special cell biological questions which can be solved with the cell culture model according to the invention are, for example:

• 1. Treatment of epithelial cells with drugs / pollutants / inflammatory mediators directly on the basolateral and / or apical side.
• 2. Evidence of the efficacy of drugs / pollutants / inflammatory mediators on the epithelial barrier
• 3. Investigation of the growth-stimulating and growth-inhibiting effects of drugs.
• 4. Analysis of released cell waste products during appropriate experiments.
• 5. Cellular interactions by cocultivation of different cell types, e.g. B. with fibroblasts or neutrophils granulocytes.
• 6. Attachment behavior of epithelial cells on modified collagen matrix, wherein the composition can be adapted analogous to the extracellular matrix in inflammatory airways.

The invention will be explained in more detail in 3 embodiments.

Embodiment 1

Creation of a cell line

For the present invention, primary bronchial epithelial cells from horses are used which are enzymatically recovered from the tissue immediately after killing the animals. For this purpose, the epithelial layer is manually detached from the bronchi under sterile conditions, washed in HBSS and minced. Subsequently, the tissue in Erlenmeyer flasks is mixed with 0.25% trypsin / EDTA solution and digested at 37 ° C. and 5% CO ₂ . After two hours of enzymatic digestion, the reaction is stopped by adding 20% fetal bovine serum (FBS) in HBSS. After several pacifiers, the tissue suspension is filtered with a pipette through sterile gauze filters and 40 μm cell sieves. After centrifugation at 1500 rpm and 4 ° C for 10 minutes, the cell pellets in Airway Epithelial Cell Growth Medium (AECGM; Promocell) with 10% FBS, 200 U / ml penicillin, 0.2 mg / ml streptomycin and 5 mg / ml amphotericin B resuspended and seeded at a density of 10 ⁶ cells / cm ² . The average cell yield is 11 × 10 ⁶ cells per 500 mg of tissue with a vitality of> 95% and a purity of the epithelial cells of> 92%.

Embodiment 2

Culture of EBEC on culture plates / bottles

Culturing of the EBEC on tissue culture plates / bottles is carried out using the culture medium AECGM with 10% FBS in the presence of 200 U / ml penicillin, 0.2 mg / ml streptomycin and 5 mg / ml amphotericin B. Particularly advantageous for the attachment, the Proliferation and growth of the EBEC cell line is in this case a coating of the growth surface with 10 μg / cm ^{2 of} collagen 1. After seeding of 10 ⁶ cells / cm ² , a confluent cell lawn develops with a cobblestone-like appearance within one week. The cultured cells correspond in their morphology and protein expression to the cells of origin and express the epithelial cell marker cytokeratin. In addition, they increasingly form tight junctions with increasing culture time, which is characterized by an accumulation of zonular occludens 1 protein in the cell membrane. Due to the stable proliferation of the EBEC under the described culture conditions ( 1 ), the cells can be subcultured to collagen 1-coated culture plates using 0.05% trypsin / EDTA.

Embodiment 3

Culture of EBEC on semipermeable membrane

In the preferred embodiment of the invention the isolated equine bronchial epithelial cells are coated on a 20 g / cm ² Collagen type 1, cult fourth semi-permeable membrane having a pore size of 0.4 .mu.M in 24-well plates. Alternatively, it is also conceivable to cultivate the cells on larger cell culture inserts in 12-hole or 6-hole culture plates. The culture of the EBEC cell line in the inserts allows for separate access to either the apical or basal side of the barrier, and is excellent for assessing cells by light microscopy and immunocytochemistry. Cultivation is carried out first submerged using the above culture medium AECGM with 10% FBS in the presence of 200 U / ml penicillin, 0.2 mg / ml streptomycin and 5 mg / ml amphotericin B. Grow after seeding 10 ⁶ cells / cm ² EBEC within a week to a confluent cobblestone-like cell lawn with functional tight junctions. As a measure of cell differentiation and barrier function, the transepithelial electrical resistance (TEER) is measured. When the resistance reaches a threshold of 200 Ω / cm ² , it switches to the air-liquid (ALI) culture condition with the liquid removed from the apical compartment of the insert and the AECGM in the basolateral compartment removed by DMEM / Ham's F-12 with L- glutamine, 2% Ultroser ^® -G and 15 ng / ml retinoic acid is replaced. Within 10 days, the EBEC under ALI conditions differentiate into a polarized, multiseriate, functional epithelium from mug and ciliated cells with stable expression of the tight junction protein Zonula-Occludens-1. 2 shows a typical course of TEER formation of EBEC under ALI culture conditions as an expression of the integrity of the epithelial layer.

The EBEC cell line cultured on inserts can be used to study the differentiation of airway epithelium and barrier function as well as the influence of substances such as inflammatory mediators, drugs or potentially toxic substances on the epithelial barrier. Thus, in contrast to existing culture models with equine respiratory epithelial cells via the TEER, the formation of the functional barrier can be quantified in the present invention.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• DE 102004024834 A1 [0003]
• CA 2508256 [0003]
• CA 2445961 A1 [0003]
• CN 102787095 [0003]

Claims (7)

Cell line of horses tissue, characterized in that it comprises epithelial cells from the bronchi and is available under conventional adhesion culture and in the air-liquid interface. A cell line according to claim 1 obtainable by expressing epithelial cytokeratin and zonula occludens-1 of tight junctions. A cell line according to any one of claims 1-2, obtainable by dissolving epithelial cells from the bronchi of horses using 0.25% trypsin from mechanically detached mucosa pieces and filtering the digested cells through 40 μm mesh cell sieves. A cell line according to any one of claims 1-3, obtainable by culturing the isolated epithelial cells in adherent culture using AECGM with 10% FBS on collagen-coated culture plates. A cell line according to any one of claims 1-3, obtainable by culturing the isolated epithelial cells in adherent culture using AECGM with 10% FBS on collagen-coated Transwell inserts and after formation of an intact monolayer using 2% DMEM Ham's F12. Ultroser and retinoic acid are cultivated in the air-liquid interface. A cell line according to claims 1, 2, 3 and 5 obtainable by having a transepithelial electrical resistance of at least 200 Ω / cm ² . Cell line according to the preceding claims, characterized in that it can be used to study pathogenetic processes of respiratory diseases and to analyze the effect of drugs and toxicological effects of substances.

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