[Preparation of the amniotic membrane with the waterjet].
BACKGROUND
Ex vivo culturing of limbal stem cells on human amniotic membranes can be accelerated if all amniotic epithelial cells have been removed beforehand. A common application of acellular amniotic membranes is their use in cultivating autologous oral mucosal epithelial cells for transplantation in cases of bilateral stem cell insufficiency. Amniotic epithelial cells can be eliminated with enzymatic-chemical or mechanical methods or with a combination of both.


METHODS
The efficacy of a waterjet cutter to eliminate amniotic epithelial cells from the amniotic membrane was investigated. Deep frozen placentas from healthy mothers were defrosted and a well-defined surface of the amniotic membrane (d = 15 mm) was treated with the waterjet in a standardized way. The waterjet used two different nozzles (pin-point and narrow stream nozzles). The applied system pressures with the pin-point stream nozzle (aperture 120 μm) were 30, 40 and 50 bar and the narrow stream nozzle was operated with pressures of 70, 80 and 90 bar on the amniotic membrane. A total number of 42 tissue samples were examined with an optical microscope using native trypan blue staining. For each type of nozzle and each application pressure two amnion samples were examined with a scanning electron microscope to analyze the efficacy of the mechanical epithelial cell elimination from the amniotic membrane. After medical imaging and histopathological examination the efficacy was graded using the following scale: 0 = no amniotic epithelial cells, 1 = no cells, low amounts of cell debris, 2 = single amniotic epithelial cells, large amounts of cell debris, 3 = loose cell layer, 4 = continuous sheet of epithelial cells.


RESULTS
To eliminate epithelial cells from the surface of the amniotic membrane with the waterjet pinpoint stream nozzle (aperture: 120 μm) an application pressure of 30-50 bar was needed. The use of the narrow stream nozzle required a pressure of 70-90 bar.


CONCLUSIONS
The preparation of amniotic membranes with the waterjet represents a precise option to mechanically eliminate amniotic epithelial cells from the amniotic membrane. The use of a waterjet cutter as an exclusively mechanical method without enzymatic-chemical substances may be a benefit, as cytotoxic effects on culturing limbal stem cells caused by chemical substances are not present.
Universitäts-Augenklinik, Ernst-Moritz-Arndt Universität, Ferdinand-Sauerbruch-Str., 17475, Greifswald, Deutschland.
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P
Schreier
J
J
Darmann
C
C
Jürgens
R
R
Schlüter
J
J
Giebel
F
F
Tost
D000328:Adult; D000650:Amnion / Q000166:cytology*; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D016039:Corneal Transplantation; D004210:Dissection / Q000379:methods*; D004847:Epithelial Cells / Q000166:cytology*; D005260:Female; D006801:Humans; D033581:Stem Cell Transplantation / Q000379:methods*; D013234:Stem Cells / Q000166:cytology*; D013499:Surface Properties; D055815:Young Adult