DE19513177A1 - Cell layer prepn. and transport system for use in skin transplants - Google Patents

Abstract

Substrate cell layers containing a new collagen- or gel-type carrier as well as amnion or acellular dermis as substrate and a layer of cultured cells. Also claimed are (1) a method of preparing amnion preparations consisting of allogenic or xenogenic amnions which have been sepd. from the placenta and cleaned of attached substances and chorion parts; (2) a method for preparing above mentioned substrate cell layers, and (3) a transport system for the above mentioned preparations comprising an outer unbreakable coat and an inner chamber with loose sepg.-floors, for the storage of the cell layers, fixed by stamps, culture medium, heat sources for the temp. control and a controllable CO2 supply.

Description

The invention relates to novel Amnionpreparate, process for their preparation and their Use for human medicine, eg. B. as a skin replacement material or as a carrier for multilayer cell layers.

Embryonal hulls, also termed haysticks, fruit hulls or seed hulls, represent of animal and human embryo derived cell aggregates representing the body envelop. They rupture before or at birth. The embryo covers consist of the inner (amnion) and outer (chorion) sheath. The amniotic cavity is with Amniotic fluid filled for the protection of the embryo (Lexikon der Biologie, Spektrum Academic Publishing Heidelberg - Berlin - Oxford, 1994).

Amniæ have been used as wound dressing materials for many years, in particular as temporary skin replacement in burn wounds, in particular Human amnion membranes (eg, Kucan, J.O., et al., Ann-Plast-Surg., 1982, 8 (6): 523- 527; Lin, SiD. et al., Burns-Incl-Therm-Inj. 1985, 11 (5): 374-378; Gobel, P. et al. Orthop-traumatol. 1990, 37 (9): 495-498), but also in general surgery, e.g. B. as a wound dressing in chronic ulcerations (Shun, A. et al., Med-J-Aust. 1983, 2 (6): 279-283), in orthopedics for preventing infections (Vishwakarma, G.K. et al. J-Bone Joint Surg Br. 1986, 68 (1): 68-74) or in reconstructive surgery (Lawson, V.G., Aust-N-Z-Y-Surg., 1986, 56 (2): 163-166; Zohar, Y., et al., Laryngoscope, Vol. 1987, 97 (8 Pt 1): 978-980; Fishman, I.J. et al., J-Urol. 1987, 138 (5): 1291-4).

The use of the human placenta as the starting material for the extraction of implants for vascular and skin surgery is the subject of DE-OS (German Laid-Open) 3608329 (Bunge, J., 1987). This is also the Amnion as suitable skin graft for covering large burns (see also DE-OS 32 40 909, Fauck, W.P., 1983). Amnion membranes are also as a substrate tissue for evaluating the growth of cells as well as Growth substrate for cell cultures has been described (US-PS (patent specification) 4446234, Russo, R. et al. 1984).

It is also known that a preferred method for the storage of Skin grafts, e.g. In the European Skin Bank, is donor skin in  To preserve glycerol (de Backere, A.C.J .: "EuTO Skin Bank: large scale skin banking in Europe based on glycerol preservation of doner skin ", Burns 1994 20 (1) 4-9; Hettich R. et al .: "The immunogenicity of glycerol-preserved doner skin", Burn 1994, 20 (1) 71-76. Recently, a new protein called kalinin has become known the adhesion of transplanted keratinocytes to a substrate pad; a. also Amnion, (U.S. Patent No. 5,352,668, Burgeson, R.E., 1994).

The invention has for its object to provide amniotic preparations, which are in a usable condition for stockpiling. The task was solved by separating embryonic envelopes from the placenta, from freed of adhering components, the chorion strips off in a moist state and the remaining amniotic fluid in preserving solutions. use find embryonic envelopes after spontaneous births. Put human amniotic preparations Donors advance, who agree to a survey on possible Exclusion criteria and for infection serology examinations. In In this case, the embryonic envelopes are frozen and allowed to release intermediately stored negative infection serological findings. The stripping of the chorion takes place on a disinfectable solid base. After sterilization of cut amnesia with a peracetic acid-alcohol mixture and renewed The preparations are washed in preservative solvents with cooling kept.

It has been found that the Amnionpräparate invention good show skin-friendly properties and as a carrier material for cell types that over time of cell growth form multilayered sheets, such as keratinocytes are suitable.

The invention will be explained in more detail with reference to embodiments.

embodiments example 1 General rule

The delivery of the embryonic envelopes in frozen state with serum in airtight sealed bags of containers. The storage temperature is up to for processing -20 to -40 ° C. For processing, the material is in a suitable Thawed vessel in cold to lukewarm water. After thawing becomes one Water change made.  

In this water bath, the material is shaken in glasses with screw cap, (about 3 to 5 minutes). This involves the dissolution of blood and other attached Tissue components as well as the partial separation of Amnion and Chorion.

Then the material is placed on a smooth Präparierunterlage (disinfectable) and By blunt loosening Amnion separated from the chorion (chorion may be discarded).

After straightening the tears with the scissors (to prevent further tearing) prevent), the area is measured, marked by a control mark and the prepared amnion with the control mark in a special container for amnion and skin (The vessel is through special openings flooded for the different Media).

After subsequent rinsing under running water, the sterilization process takes place in a vacuum (desiccator) with shaking for 2 hours, taking out the sterilization solution

Glycerol | 375 ml Wofasteril 19 ml ethanol 250 ml Distilled water 356 ml

consists.

After the sterilization time, the further operations are carried out under Clean room conditions (Class A), such as rinsing in a mixture of sterilized isotonic saline and sterilized glycerin.

Thereafter, the removal takes place from the o.g. Special rinsing vessel and quality control of the Amnion preparation, sterile sampling and packaging (final packaging with transfer to sterilized glycerol and definitive occlusion).

Example 2 Human amniotic preparation 2.1. withdrawal

The removal of the embryonic envelopes from the abdominal (placenta) happens on the legal basis of the written consent of the donor, the pharmaceutical relevant donor exclusion criteria and the written declaration (removal protocol) of the treating physician of the suitability of the tissue for the production of a clinically usable without hesitation.

2.2. Temporary storage and transport

Intermediate preservation takes place in the clinic

• - in a bottle with saturated saline and refrigerator conditions or  
• - sealed in a plastic bag and frozen at -20 ° C or below or
• - by simple storage in physiological saline and normothermic storage

or closure in plastic bag and normothermic storage when picking up within 12 hours of collection.

The transport takes place in an insulating box under cooling conditions. At removal and Transport within 12 hours after removal, transport without additional cooling done. Exceptions are ambient temperatures above 20 ° C.

2.3. Interim storage until preparation

After delivery of the Amnion source material from the "collection site" (obstetric Facilities), the tissue remains in quarantine storage (-18 ° C or below) until on the presence of infection serological findings (AIDS, hepatitis, lues, Cytomegalovirus). This intermediate storage will be until the beginning of the mechanical Processing expanded.

2.4. Mechanical preparation

In preparation for the mechanical preparation, the tissue is placed in a water bath thawed. This is followed by a washing process to remove adhering tissues and of blood.

In this water bath, the fabric is in glasses or plastic bottles with Screw cap shaken for about 3 to 5 minutes. The easier release takes place of blood as well as the already partial separation of Amnion and Chorion.

On a disinfectable solid surface then the amnion is in a moist state freed from chorion by stripping.

This is followed by a straightening of any break points with scissors or others mechanical cutting instruments. This will further tearing and Becoming unusable of the preparation. The remaining area becomes measured. The product is labeled with a mark for identification.

2.5. sterilization

The thus treated and purified Amnion is now a sterilization process subjected.  

This sterilization is carried out with a peracetic acid-alcohol mixture. This will be a Special vessel that uses both the mixing of individual amniotic preparations prevents as well as through specially placed openings easily with the used Solutions can be flooded (flowed through).

The solution preferably consists of:

- Glycerol | 375 ml - Wofasteril 19 ml - ethanol 250 ml - Aqua dist. 356 ml

The sterilization process is preferably carried out over a period of 2 hours Shaking done.

Sterilization is performed by rinsing to wash out the sterilization solution with (preferably) isotonic sterile saline and sterile glycerol. After sterilization, all further operations are carried out under clean room conditions (cleanroom class A).

2.6. sterile control

The next step is to remove the amnion from the o.g. Special vessels and the removal of a sample for sterile control.

2.7. packing

The sterilized amnion is now in a definitively occlusive vessel, preferably from Glass or plastic filled with sterile glycerin. this is the Final packaging.

2.8. Documentation, label according to drug law 2.9. Disposal / deposit attitude

Until delivery to the purchaser, the amniotic preparation is at refrigerator temperature or stored underneath.

The Glycerinkonservierung also allows a damage-free storage at Zero temperatures. This means an extension of the durability and thus Extension of storage time.  

Example 3 Bovine amniotic preparation (flow chart)

The embryonic envelopes are separated from the placenta after spontaneous delivery, frozen and stored at -18 ° C until released by negative infection serology Intermediate findings.

For further processing, the amniotic fluid is thawed in a water bath.

For the removal of blood u. a. adherent components are the embryonic envelopes with Washed water.

On a disinfectable solid surface, they are in a wet state Stripping freed from chorion. The chorion is discarded.

The remaining Amnion is again rinsed with water and then on the tailored size.

Sterilization is carried out with 1% peracetic acid with the addition of alcohol and glycerol shaking for 2 hours.

This is followed by washing out the sterilization solution with isotonic saline solution and the removal of the sterility control.

The following are: Preparation of the preparation under Laminar Air Flow (cleanroom class A) in definitely closable vessels in glycerin.

Store at refrigerator temperature or below.

Delivery of the preparation on request.

Example 4 support material

Cell types that form multilayer cell layers in the course of cell growth, eg. B. Keratinocytes are in culture vessels on feeder cells or with basement membrane bred proteins coated cell culture surfaces, the cell layers enzymatically detached and placed on different substrates.

The amniotic preparations according to the invention are advantageously suitable for Use of such carrier materials.

Claims (7)

1. Amniotic preparations, consisting of amniotic origin, separated from the placenta Embryonic envelopes after removal of adherent components and chorion contents emerge. 2. Amnion preparations according to claim 1, characterized in that they are human or represent bovine amnion preparations. 3. amnion preparations according to claim 1 and 2, characterized in that the Embryonic sheaths separated from the placenta after spontaneous delivery, deep-frozen stored, thawed after the infection serological release, of adherent blood and tissue freed, in a moist state, the chorion stripped, the Tailored amniotic, sterilized, washed, preserved Solution and stored cool after assembly. 4. A process for the preparation of Amnionpräparaten, characterized in that the embryonic envelopes are separated from the placenta after spontaneous delivery, frozen stored, thawed after the infection serological release, of adherent blood and tissue freed, in a moist state, the chorion stripped, the Tailored amniotic, sterilized, washed, preserved Solution and stored cool after assembly. 5. The method according to claim 4, characterized in that the embryonic envelopes in Temperatures of at least -10 ° C frozen stored, the stripping of the Chorions on a disinfectable solid underlay done with a sterilization Peracetic acid-alcohol mixture, the washing process with isotonic sterile Saline and sterile glycerin and the final packaging in sterile glycerin is made. 6. Use of the Amnionpräparate according to claim 1 to 5, characterized that they as a skin substitute material or as a carrier material for multilayer cell layers be used.   7. Use according to claim 6, characterized in that the use as Skin replacement material, especially for burn wounds and as a carrier material especially for keratinocytes.