Gelatin  glutaralgehyde modified vitelline membrane of hen's egg supports growth of adherent cells in vitro.
The vitelline membrane of the hen's egg (VMHE) is transparent fibrous bilayer which encloses the yolk. The inner layer (lamina perivitellina) laid down around the yolk while it is still in the ovary, whilst the outer layer (lamina exravitellina) is added after ovulation as the yolk passes down the oviduct [1-2]. In the inner layer there are at least four different glycoproteins (GPI-GPIV). The outer layer consists mainly of lysozyme, ovomycin, lectin and two other proteins--the vitelline membrane outer proteins (VMO1 and VMO2).
It was observed that both membrane layers differ in thickness . The outer one varies from 3.0 to 8.5 and the inner one from 1.0 to 3.5 [micro]m. With an indication for high individual variations, the weight of the freeze-dried VMHE of a fresh egg was found to be 6.79 mg (5.23 mg  10.51 mg) and the protein percentage of the total membrane weight  70% (58.9%-76.0%) . The VMHE strength evaluated by texture analyzer amount to 577.10 or 463.40 (TA force/g) depending on the hen's breed .
During the egg storage at room temperature most of the physical and biochemical properties of the VMHE undergo changes that could be summarized as follows: loss of integrity manifested morphologically by disappearance of fibrous structure, particularly of the outer layer; lessening of the membrane strength due to rise in pH of the albumen from about 7.8 (fresh laid egg) to about 9.5 after 21 days storage at 20[degrees]C or 14 days at 30[degrees]C, disintegration of proteins VMO1 and VMO2 from the membrane  and degradation of the major structural glycoprotein II of the inner layer ; increase of the lysozyme content of VMHE.
VMHE is not used as a substrate in cell culture techniques, most likely because it undergoes rapid deterioration at temperatures from 20[degrees]C to 37[degrees]C and by reason of its high lysozyme content  55% of the total protein content of VMHE (fresh egg) and 70% after 30 days storage of the egg at 20[degrees]C . It is well-documented fact that lysozyme possesses not only lytic activity against Gram-positive bacteria but also against some eukaryotic cells . Furthermore lectins inhibit the proliferation of different cells in vitro including breast cancer cells . It has been already mentioned that lectin is present in the outer layer of VMHE .
In accordance with the data indicated above, the aim of the present study was to determine the capacity of VMHE isolated from fresh egg (VMHE-f) and gelatin  glutaraldehyde modified VMHE (VMHE-m) to support growth of adherent cells in vitro. Light microscopic observations were used for assessment of the cell behavior at different culture conditions.
Materials and Methods
Isolation of vitelline membrane
Fresh eggs (weight 60  65 g., laid within last 24h) were obtained from flock of White Leghorn hens of the same age group. After egg-breaking the yolk was separated from as much albumen as possible and then submerged in PBS pH 7.2. An incision allowed detachment of the VMHE as the yolk ran out into the buffer. The isolated VMHE was cut in large pieces and washed in PBS at room temperature by gentle shacking on plate shaker (Titertec Flow Lab[R]) until it become fully transparent free from adherent yolk. The washed VMHE was kept in PBS at 4[degrees]C until use.
Modification and gelatin coating of the vitelline membrane by glutaraldehyde cross-linking
Glutaraldehyde treatment of VMHE was performed in two different ways:
a) One-step procedure: Pieces of washed VMHE-f were soaked for 1h in a vessel containing 30 ml 5% w/v gelatin in PBS. At continuous shake, a solution of 30 ml 5% v/v glutaraldehyde in PBS was added in small portions for 1h at room temperature. By means of forceps the pieces of gelatin-glutaraldehyde modified membrane (VMHE-m) were placed consecutively into other vessels, washed several times in PBS and kept in DMEM at 4[degrees]C
b) Two-steps procedure: Washed pieces of VMHE-f were soaked and shaken for 1h at room temperature in a vessel containing 30 ml 2.5% v/v glutaraldehyde in PBS. Glutaraldehyde treated membranes were washed several times in PBS, transferred and shaken for 1h in a vessel containing 30 ml 5% w/v gelatin in PBS. After washing in PBS the pieces of VMHE-m were immersed in DMEM and kept at 4[degrees]C.
Stretching of the membrane pieces
Small pieces of VMHE-m and VMHE-f (approx. size 1x1 cm) were placed wet on standard (20x20 mm) sterile coverslips and carefully stretched under stereo microscope using eye forceps. The coverslips were then moved into polystyrene Perti dishes (35x10 mm) and allowed to dry under UV light for overnight period.
Isolation, modification and stretching of VMHE were done under sterile conditions.
Adherent cell cultures and light microscopic observations
In order to examine the capacity of fresh and modified vitelline membrane to support growth of adherent cells in vitro we made use of three different cell types  3T3 cells (ATCC CCL-92), HeLa cells (UKKK.ATCC CCL2) and macrophages collected by peritoneal lavage from BALB/ c mice according to the recommendation of (Stuart et al., 1973). The cells were seeded in Petri dishes containing coverslips with stretched pieces of VMHE-f or VMHE-m at densities: for 3T3 cells  [2.10.sup.4] cells/dish, for HeLa cells  [2.10.sup.5] cells/ml and for mouse peritoneal macrophages [1.10.sup.6] cells/ml (including non-adherent peritoneal cells). All cultures were allowed to grow for 3 days in 2ml/dish culture medium DMEM (Sigma[R]) supplemented by 10% fetal calf serum (Sigma[R]), penicillin 100U/ml and streptomycin 100 Mg/ml, at 37[degrees]C, 5% C[O.sub.2] in humidified C[O.sub.2] incubator. Four hours after incubation onset the non-adherent mouse peritoneal cells were removed by washing and the Petri dishes were filled anew with 2 ml culture medium . By inverted microscope the growth of the cell cultures were examined ones daily from day 1 to day 3. Cytological preparations from the adherent cell cultures (day 1 and day 3) were made. For the purpose, the coverslips were washed twice in PBS and fixed for 10 min at room temperature in 3.7% w/v formaldehyde in PBS. After washing in PBS the coverslips were treated for 1 min with cold methanol (-20[degrees]C), fan dried for 15 min, washed in distillate water and stained according to the standard hematoxylin/eosin (H/E) protocol. The coverslips were mounted cell-side down on microscope slides using a solution of glycerol-PBS (9:1) as a mountant. The slide observations were performed on standard upright light microscope equipped with CCD camera. The cells were characterized based on their morphology (shape, appearance, presence of mitotic figures and cell confluence). Because the glass (Si[O.sub.2]) is among the best substrates for adherent cell culture, we made observations at the edges of the stretched VMHE pieces where morphological difference between cells growing on VMHE and on glass could be found.
Results and Discussion
The light microscopic observation revealed that all the cell types used in our experiment showed absence of adherence to the pieces of VMHE-f stretched on the coverslips. In Petri dishes containing 3T3 cells seeded on VMHE-f, an atypical growth pattern manifested by formation of semi-adherent 3D clusters of round shaped cells was observed.
Growth of 3T3 cells on VMHE-m
During the cultivation 3T3 cells showed morphological characteristics typical for the active fibroblasts growing in vitro  spreading, strong attachment to the substrate, polygonal shape, branched cytoplasm and high nuclear: cytoplasmic ratio. On day 3 the cells covered entirely the VMHE-m pieces forming confluent ordered monolayer (Fig.I. (B)). No detectable morphological difference was observed between cells growing on VMHE-m and on glass surface (Fig.I. (A)). In cell preparations VMHE appeared not so transparent because of its affinity towards eosin stain.
Growth of HeLa cells on VMHE-m
Like 3T3 cells, HeLa cells showed good attachment and spreading on VMHE-m. Identical morphological characteristics (round to elongated shape, lamelliopodia formation on the leading edge of the cells and reaching to sub-confluence at the end of the incubation period) were noted for cells attached to VMHE-m or to glass surface (Fig.I. (C),(D)).
Growth of peritoneal macrophages on VMHE-m
In contrast to 3T3 cells and HeLa cells, mouse peritoneal macrophages are highly differentiated cells taking part in non-specific immune defense of mammals. In general macrophages are adherent cells that exhibit two basic types of morphology in culture: there are either flat and irregular in shape with thin cytoplasmic extensions or rounded with abundant sytoplasm. It was found that the proportion of the flat cells increases with increasing time in culture. In young cultures the rounded forms are predominant . In our experiment we found that mouse peritoneal macrophages adhere to VMHE-m as firmly as they do that to the glass surface. On day 3, we observed flat (spread) and round forms to be present attached to VMHE-m. Some of round shaped cells were in mitotic state (Fig.I (E),(F)).
[FIGURE 1 OMITTED]
Glutaraldehyde is an amine-amine crosslinker frequently used for immobilization of enzymes  or hydrophilic coatings on many biomaterials. The hydrophilic coatings especially those containing soluble proteins of the extra-cellular matrix improve biocompatibility and/or cytocompatibility of bio-surfaces and afford an opportunity for their application in design of medical implants. Hua A.I. et al. succeeded in achieving endothelial cell adhesion and growth on silicone rubber by means of gelatin-glutaraldehyde cross-linking of this hydrophobic material . Glutaraldehyde has been used in variety of applications where the maintenance of the structural rigidity of the proteins is important. By glutaraldehyde cross-linking, Spoerl et al. attained significant increase of biomechanical strength, enzymatic resistance, transparency and less wrinkling of amnion used for surface reconstruction of the cornea. Glutaraldehyde treated membranes were completely resistant to enzymatic digestion (performed by 0.1% collagenase solution), while fresh and cryopreserved amnions were dissolved by day 7. In patients the cross-linked membrane was preserved for up to 90 days without any signs of dissolution .
Using morphological criteria we evaluated the capacity of VMHE to support growth of adherent cells in vitro. The preliminary results clearly show that the utilized cross-linking procedures turn fresh and non-cytocompatible VMHE into a good culture substrate for adherent cell lines and for differentiated adherent cells. With its inherent properties  transparency, flexibility, avascularity and relatively small thickness, vitelline membrane of the hen's egg and VMHE-m in particular could find application in design of membrane-cell-grafts for clinical use. In reference to immunogenicity of VMHE-m can be concluded that the gelatin, a well known non-immunogenic biomaterial [13-14], probably veils the determinants of the membrane antigens. The absence of morphological difference between mouse peritoneal macrophages growing on VMHE-m and on glass surface is a preliminary indication in favor of this assumption.
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Marin Bratanov, Anatoliy Neronov *, Ilina Vavrek, Elena Nikolova
Institute of Experimental Morphology and Anthropology with Museum. Bulgarian Academy of Sciences. Acad. G.Bonchev bl.25, Sofia 1113, Bulgaria
Corresponding author (firstname.lastname@example.org) Anatoliy Neronov
Received 3 August 2010; Accepted 3 August 2010; Available online 1 March 2011
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|Title Annotation:||Short Communication|
|Author:||Bratanov, Marin; Neronov, Anatoliy; Vavrek, Ilina; Nikolova, Elena|
|Publication:||Trends in Biomaterials and Artificial Organs|
|Date:||Jan 1, 2011|
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