Punch and spindle-shaped biopsies for collecting oral mucosal tissue for the fabrication of transplantable autologous epithelial cell sheets

The oral mucosa is an easily accessible source of cells. Oral mucosal collection will be an essential surgical procedure for regenerative medicine and cell biological research. However, there is no current report that describes the details of the surgical procedure used for oral mucosal collection. Moreover, the number of cells that can be obtained has not been determined. Two different procedures, the punch biopsy and the spindle-shaped biopsy, were performed for the fabrication of transplantable autologous epithelial cell sheets. The mean values of the cells collected per square centimeter of tissue using the punch biopsy and the spindle-shaped biopsy were 76.8 ± 45 × 10(4) cells/cm(2) and 195.7 ± 120 × 10(4) cells/cm(2) , respectively. There was no significant difference between the punch biopsy and the spindle-shaped biopsy. The coefficient of variation of the punch biopsy and the spindle-shaped biopsy was 58.9% and 69.8%, respectively. This result indicated that both procedures showed variations in the number of collected cells. Although the punch biopsy may be easier and simpler than the spindle-shaped biopsy, multiple punch biopsies may result in a more complicated procedure, and the spindle-shaped biopsy may be preferable when a large number of cells is necessary. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A:2849-2854, 2012.     

Fabrication of transplantable human oral mucosal epithelial cell sheets using temperature-responsive culture inserts without feeder layer cells

To exclude bacteria- or animal-derived factors from cultured fabrication of transplantable epithelial cell sheets, primary human oral mucosal epithelial cells were seeded on temperature-responsive culture inserts having submicron-scale pores. Supplying culture medium containing human autologous serum to both apical and basal sides of human epithelial cells allows these cells to grow to confluence. These proliferating cells created stratified epithelial layers even when 3T3 feeder layers and fetal bovine serum were eliminated from culture. Normal keratin expression profiles were obtained with these cells, and basal and midlayer cells expressed p63, a putative stem/progenitor marker. These results suggest that temperature-responsive culture inserts can be useful in clinical settings that require the exclusion of xenogeneic factors.     

Adhesion of corneal epithelial cells to cell adhesion peptide modified pHEMA surfaces

Epithelialization of a corneal implant is a desirable property. In this study we compared surface modification of poly (2-hydroxyethyl methacrylate) (pHEMA) with the cell adhesion peptides RGDS and YIGSR. Various parameters in the tresyl chloride activation and modification reactions were considered in order to maximize surface coverage with the peptide including tresyl chloride reaction solvent. tresyl chloride reaction time, tresyl chloride concentration, peptide concentration, and peptide reaction pH. Surface chemistry and corneal epithelial cell adhesion to the modified surfaces were examined. X-ray photoelectron spectroscopy data suggested that while peptide modification had occurred, surface coverage with the peptide was incomplete. Acetone was found to result in a higher fraction of nitrogen and surface bound carboxyl groups compared to dioxane and ether. Furthermore, corneal epithelial cell adhesion to the surfaces for which acetone was used for the activation reaction was significantly greater. Statistical analysis of the various samples suggests that lower peptide concentrations and higher tresyl chloride reaction times result in better cell adhesion. Furthermore, modification with YIGSR resulted in higher surface concentrations and better cell adhesion than modification with RGDS. Little or no cell adhesion was noted on the unmodified pHEMA controls. Protein adsorption results suggest that the differences in cell adhesion cannot be attributed to differences in serum protein adsorption from the culture medium. We conclude that YIGSR modified surfaces have significant potential for further development in corneal applications.     

Production of genetically modified cells expressing specific transgenes by retroviral vectors for gene therapy

Summary Techniques and protocols are described for the generation of genetically modified cells that can be used for gene therapy. Primary fibroblast cultures are established from skin biopsies, maintained in culture, frozen for long-term storage, and retrieved when necessary. Retroviral packaging cell lines are generated by transfection of DNA into retroviral packaging cells by calcium-phosphate precipitation method or by lipofection method. To generate cell lines expressing high titer virus, individual colonies of cells are cloned and the virus titer is determined. Virus collected from packaging cells expressing high titer virus is then used to infect primary fibroblasts. To obtain fibroblast cell lines expressing high amounts of transgenes, individual cells can be cloned to generate clonal cell lines. Although the methods described here are for fibroblasts, the same methods or modification of the methods can be used for other cell types.     

Corneal regeneration by transplantation of corneal epithelial cell sheets fabricated with automated cell culture system in rabbit model

We have performed clinical applications of cell sheet-based regenerative medicine with human patients in several fields. In order to achieve the mass production of transplantable cell sheets, we have developed automated cell culture systems. Here, we report an automated robotic system utilizing a cell culture vessel, cell cartridge. The cell cartridge had two rooms for epithelial cells and feeder layer cells separating by porous membrane on which a temperature-responsive polymer was covalently immobilized. After pouring cells into this robotic system, cell seeding, medium change, and microscopic examination during culture were automatically performed according to the computer program. Transplantable corneal epithelial cell sheets were successfully fabricated in cell cartridges with this robotic system. Then, fabricated cell sheets were transplanted onto ocular surfaces of rabbit limbal epithelial stem cell deficiency model after 6-h transportation using a portable homothermal container to keep inner temperature at 36 °C. Within one week after transplantation, normal corneal epithelium was successfully regenerated. This automatic cell culture system would be useful for industrialization of tissue-engineered products for regenerative medicine.     

Interactions of corneal epithelial cells and surfaces modified with cell adhesion peptide combinations

In order to facilitate the adhesion of corneal epithelial cells to a poly dimethyl siloxane (PDMS) substrate ultimately for the development of a synthetic keratoprosthesis, PDMS surfaces were modified by covalent attachment of combinations of cell adhesion and synergistic peptides derived from laminin and fibronectin. Peptides studied included YIGSR and its synergistic peptide PDSGR from laminin and the fibronectin derived RGDS and PHSRN. Surfaces were modified with combinations of peptides determined by an experimental design. Peptide surface densities, measured using 125-I labeled tyrosine containing analogs, were on the order of pmol/cm2. Surface density varied as a linear function of peptide concentration in the reaction solution, and was different for the different peptides examined. The lowest surface density at all solution fractions was obtained with GYRGDS, while the highest density was consistently obtained with GYPDSGR. These results provide evidence that the surfaces were modified with multiple peptides. Water contact angles and XPS results provided additional evidence for differences in the chemical composition of the various surfaces. Significant differences in the adhesion of human corneal epithelial cells to the modified surfaces were noted. Statistical analysis of the experimental adhesion results suggested that solution concentration YIGSR, RGDS, and PHSRN as well as the interaction effect of YIGSR and PDSGR had a significant effect on cell interactions. Modification with multiple peptides resulted in greater adhesion than modification with single peptides only. Surface modification with a control peptide PPSRN in place of PHSRN resulted in a decrease in cell adhesion in virtually all cases. These results suggest that surface modification with appropriate combinations of cell adhesion peptides and synergistic peptides may result in improved cell surface interactions.     

Transplantable retinal pigment epithelial cell sheets for tissue engineering

The native retinal pigment epithelium (RPE) exists as a monolayer structure and is critically involved in the maintenance of photoreceptors. Damage or destruction of the RPE due to a variety of diseases therefore often results in loss of vision. With regenerative purposes in mind, we have examined various culture conditions such as the initial cell density and the addition of various supplements in an effort to produce transplantable RPE cell sheets that can be harvested without defects. We demonstrate that the cell density in cultured RPE sheets increased linearly with the number of seeded cells and that RPE sheets were harvested without defects and limited contraction due to cytoskeletal reorganization, when TGF-beta2 was added to the growth medium. Results from histological analysis and the measurement of trans-epithelial resistance also demonstrates that these RPE cell sheets exist as monolayer structure, similar to the native RPE, with intact cell-to-cell junctions. Therefore, these methods provide significant insight into the fabrication of transplantable RPE cell sheets that can be applied to RPE regenerative therapies to restore lost vision.     

An alternative technique for the harvesting of cultured epithelial cell sheets

We describe an efficient and economical method for the harvesting of confluent cell sheets from tissue culture flasks. Our method eliminates the use of clips by utilizing a glass hockey stick and the adherent property of the vaseline gauze carrier to enable the cultured epithelial autograft (CEA) to be rapidly removed from the tissue culture flask.     

The effect of micropores in the surface of temperature-responsive culture inserts on the fabrication of transplantable canine oral mucosal epithelial cell sheets

Primary canine oral mucosal epithelial cells were cultured on temperature-responsive dishes and cell culture inserts to fabricate transplantable epithelial cell sheets. When 3T3 feeder layers and fetal bovine serum were eliminated from dish culture, the harvested cell sheets became significantly more fragile. In contrast, when epithelial cells were cultured on inserts having submicron-scale pores, cell sheet fragility was eliminated. Keratin expression profiles showed no differences among the harvested cell sheets, but the expression of p63, a putative stem/progenitor marker, was strongly dependent on the presence of 3T3 feeder layers and serum. These results suggest that the maintenance of stem/progenitor cells is influenced by the apical/basal supply of nutrients as well as culture supplements.     

AMEGA: antigen-mediated genetically modified cell amplification

Selection of genetically modified cells is a critical step to engineer the cells with desired properties. While antibiotic selection has been commonly used, administration of cytotoxic drugs often leads to deleterious effects not only to inert cells but also to transfected or transduced ones. To overcome this problem, a positive screening method for genetically modified cells is proposed using a pair of chimeric receptors that trigger a growth signal in response to a specific antigen. Either V(H) or V(L) region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 was fused to extracellular D2 domain of erythropoietin receptor (EpoR) and transmembrane/cytoplasmic domains of either EpoR or gp130. A model transgene, enhanced green fluorescent protein (EGFP) and the chimeric receptor genes that reconstituted functional Fv were retrovirally co-infected to interleukin (IL)-3-dependent Ba/F3 cells, followed by direct HEL selection in the absence of IL-3. Consequently, a single round of selection led to a single population of EGFP-positive cells. The detailed protocol of the method termed antigen-mediated genetically modified cell amplification (AMEGA) is described.     

Tissue engineered epithelial cell sheets for the creation of a bioartificial trachea

To successfully engineer a bioartificial tracheal replacement, it is believed that the regeneration of a functional epithelial lining is a key requirement. In the present study, rabbit tracheal epithelial cells were cultured on temperature-responsive culture dishes, under normal culture conditions at 37 degrees C. By simple temperature reduction to 20 degrees C, the cultured epithelial cells were noninvasively harvested as intact sheets, without the use of any proteolytic enzymes. Support Dacron grafts that had been subcutaneously implanted for 4 weeks to allow for host tissue and vessel infiltration were then opened, and the tracheal epithelial cell sheets were transplanted to the luminal surface without sutures. These fabricated constructs were then used as tracheal replacements, in a rabbit model. Four weeks after transplantation, results showed that the tracheal grafts were covered by a mature, pseudostratified columnar epithelium. In contrast, control constructs that did not receive cell sheet transplantation demonstrated only a thin, immature epithelium at the center of the replacement graft. These results therefore demonstrate that these tracheal epithelial cell sheets can create an epithelial lining on the luminal surface of a bioartificial trachea.     

Nanoscale topography modulates corneal epithelial cell migration

The purpose of this study was to evaluate the effect of surface topographic features that mimic the corneal epithelial basement membrane on cell migration. We used electron-beam and X-ray lithography and reactive ion etching to pattern silicon wafers with pitches (groove width plus ridge width) of nano- and microscale dimensions (pitches ranged from 400 to 4000 nm). Additionally, polyurethane patterned surfaces were created by replication molding techniques to allow for real-time imaging of migrating cells. Individual SV40-transformed human corneal epithelial cells frequently aligned with respect to the underlying surface patterns and migrated almost exclusively along grooves and ridges of all pitches. Direction of migration of individual cells on smooth surfaces was random. In cell dispersion assays, colonies of cells migrated out from initially circular zones predominantly along grooves and ridges, although there was some migration perpendicular to the ridges. On smooth surfaces, cells migrated radially, equally in all directions, maintaining circular colony shapes. We conclude that substratum features resembling the native basement membrane modulate corneal epithelial cell migration. These findings have relevance to the maintenance of corneal homeostasis and wound healing, as well as to the evolution of strategies in tissue engineering, corneal prosthesis development, and cell culture material fabrication.     

Human corneal endothelial cell sheets for transplantation: Thermo-responsive cell culture carriers to meet cell-specific requirements

Corneal endothelial diseases lead to severe vision impairment, motivating the transplantation of donor corneae or corneal endothelial lamellae, which is, however, impeded by endothelial cell loss during processing. Therefore, one prioritized aim in corneal tissue engineering is the generation of transplantable human corneal endothelial cell (HCEC) layers. Thermo-responsive cell culture carriers are widely used for non-enzymatic harvest of cell sheets. The current study presents a novel thermo-responsive carrier based on simultaneous electron beam immobilization and cross-linking of poly(vinyl methyl ether) (PVME) on polymeric surfaces, which allows one to adjust layer thickness, stiffness, switching amplitude and functionalization with bioactive molecules to meet cell type specific requirements. The efficacy of this approach for HCEC, which require elaborate cell culture conditions and are strongly adherent to the substratum, is demonstrated. The developed method may pave the way to tissue engineering of corneal endothelium and significantly improve therapeutic options.     

Development of osteogenic cell sheets for bone tissue engineering applications

The use of scaffolds in combination with osteogenic cells has been the gold standard in bone tissue engineering strategies. These strategies have, however, in many cases failed to produce the desired results due to issues such as the immunogenicity of the biomaterials used and cell necrosis at the bulk of the scaffold related to deficient oxygen and nutrients diffusion. Here, we originally propose the use of cell sheet (CS) engineering as a possible way to overcome some of these obstacles. Osteogenic CSs were fabricated by culturing rat bone marrow stromal cells in thermoresponsive culture dishes. The CSs were recovered from the dishes using a low-temperature treatment and then were implanted subcutaneously in nude mice. New bone formation was verified from day 7 post-transplantation using X-ray, microcomputed tomography, and histological analysis. The presence of a vascularized marrow was also verified in the newly formed bone after 6 weeks of transplantation. Further, osteocytes were found in this newly formed tissue, supporting the conclusion that mature bone was formed after ectopically transplanting osteogenic CSs. These results therefore confirm the great potentiality of CS engineering to be used in bone tissue engineering applications.     

Thermo-responsive poly(NiPAAm-co-DEGMA) substrates for gentle harvest of human corneal endothelial cell sheets

Gentle harvesting of corneal endothelial cell sheets grown in culture is of interest for the development of cornea replacement strategies. Thin films of a fast responding copolymer of N-isopropylacrylamide (NiPAAm) and diethyleneglycol methacrylate (DEGMA) with a phase transition temperature of 32 degrees C were prepared and evaluated for that purpose. The polymer layers were immobilized onto fluorocarbon substrates using low pressure argon plasma treatment. Cell culture and detachment experiments were performed with L929 mouse fibroblasts and human corneal endothelial cells (HCEC) at standard conditions. The hydrogel-coated supports were found to permit adhesion, spreading, and proliferation of both cell types. Harvesting of cell sheets was achieved upon lowering the temperature to about 30 degrees C. The formation of a closed monolayer as a crucial prerequisite for maintaining ionic pump function in HCEC was proven by ZO-1 immunostainung. Labeling of fibronectin indicated that the vast majority of the extracellular matrix is detached from the hydrogel coatings together with the cell layer. Inspired by this result, the reuse of the hydrogel-coated culture carriers was investigated confirming the suitability of the substrates for repeated cell harvesting. Altogether, the introduced thermoresponsive coating was found advantageous for the efficient generation of HCEC sheets and will be further utilized in transplantation strategies.     

Alginate-based microencapsulation of retinal pigment epithelial cell line for cell therapy

The goals of this study were to evaluate human retinal pigment epithelial cell line (ARPE-19) for cell encapsulation and to optimize the alginate-based microencapsulation. We used immortalized ARPE-19 cells and the transfected sub-line that expresses secreted alkaline phosphatase (SEAP) reporter enzyme. Alginate was cross-linked with different divalent cations (Ca(2+), Ba(2+), Sr(2+) and combination of Ca(2+) and Ba(2+)), coated first with poly-l-lysine (PLL), and then with alginate. Microcapsules with different pore sizes and stability were generated. The pore size of the microcapsules was assessed by the release of encapsulated fluorescein isothiocyanate (FITC)-dextrans. The viability of the cells in the microcapsules was studied in vitro by assessing the secretion rates of SEAP and oxygen consumption by the cells. The best microcapsule morphology, durability and cellular viability were obtained with alginate microcapsules that were cross-linked with Ca(2+) and Ba(2+) ions and then coated with PLL and alginate. Based on FITC-dextran release these microcapsules have porous wall that enables the rapid contents release. The ARPE-19 cells maintained viability in the Ca(2+) and Ba(2+) cross-linked microcapsules for at least 110 days. The alginate microcapsules cross-linked with Ca(2+) and Ba(2+) have sufficiently large pore size for prolonged cell viability and for the release of secreted SEAP model protein (Mw 50 kDa; radius of gyration of 3 nm). ARPE-19 cells show long-term viability and protein secretion within alginate microcapsules cross-linked with Ca(2+) and Ba(2+). This combination may be useful in cell therapy.     

Transplantation of tissue-engineered retinal pigment epithelial cell sheets in a rabbit model

The retinal pigment epithelium (RPE) plays an important role in maintaining a healthy neural retina. With changes due to age, morbidity or removal of choroidal neovascularis developed as a means ofation, damage or defects of the RPE occur. Accordingly, RPE transplantation techniques have been repairing the damaged RPE. We conducted a study to transplant tissue-engineered RPE cell sheets in a rabbit model. RPE cells were isolated from pigmented rabbit eyes and seeded on temperature-responsive culture surfaces. Cultured RPE cells were arranged as a monolayer with a cobblestone cell shape that is characteristic of native RPE. The pigmented RPE cell sheets were non-invasively harvested without enzymatic treatment simply by reducing the culture temperature. Using 3-port vitrectomy, RPE cell sheets were transplanted into the subretinal space of albino rabbits. Seven days after surgery, the rabbits were sacrificed, and the eyes were enucleated and examined under both light and electron microscopy. After transplantation, our results show that the RPE cell sheets attached to the host tissues in the subretinal space more effectively than with the injection of isolated cell suspensions. Although the cell sheets maintained a monolayer structure in most areas, they were slightly folded or wrinkled in some regions. We conclude that tissue-engineered RPE cell sheets harvested from temperature-responsive culture dishes can be effectively transplanted beneath the neural retina.     

改良组织块培养法体外培养人角膜上皮干细胞

文题释义：角膜上皮干细胞：属于单能干细胞,具有细胞周期长、低分化状态、增殖潜力大、不对称分裂等特点,定位于角膜缘基底细胞层,又称之为角膜缘干细胞,对角膜上皮细胞更新及维持角膜透明起着重要作用。角膜缘干细胞的体外培养方法：主要包括酶消化培养法和组织块培养法。酶消化培养法是利用DispaseⅡ酶破坏角膜缘上皮细胞与基底膜之间的半桥粒连接,然后剥取角膜缘上皮层,再使用胰酶将其消化为单个细胞进行培养。组织块培养法没有经过酶的双重消化,将剖取的角膜缘组织块进行贴壁,细胞游离出组织块进行贴壁生长,需要一个漫长的过程。  摘要背景：角膜上皮干细胞定位于角膜缘,又称之为角膜缘干细胞,临床上由于眼表严重热烧伤、化学性烧伤、慢性炎症等原因引起的角膜缘干细胞缺乏或功能障碍治疗较为棘手。目前利用组织工程技术体外培养角膜上皮干细胞并进行临床移植成为新型有效的治疗方向。目的：探讨在无血清培养条件下采用改良组织块培养法培养人角膜上皮干细胞的可行性。方法：人角膜缘组织来自河南省眼库,植片直径小于8 mm角膜移植术后的供者剩余眼球材料,手术显微镜下剖取角膜缘上皮层外2/3区域,采用2种方法培养人角膜上皮干细胞,常规组织块培养组是将组织块上皮面向上贴壁,加入K-SFM培养液后置于 37 ℃、体积分数为5%CO₂细胞培养箱中培养;改良组织块培养组是先将组织块浸泡于K-SFM培养液中,置于细胞培养箱中孵育12 h,然后组织块上皮面向下贴壁培养。组织块周边有细胞游离出贴壁生长记作“培养第1天”,每日相差显微镜下观察细胞生长变化。利用免疫荧光染色技术检测改良组织块培养第5,10,14天时原代细胞中p63及K3的表达。结果与结论：①改良组织块培养组出膜时间明显短于常规组织块培养组(P < 0.05),出膜率明显高于常规组织块培养组(P < 0.05);②改良组织块培养组细胞生长状态良好,培养第10天可见小体积细胞较多,聚集成灶状分布;培养第14天可见细胞克隆灶,克隆灶内细胞体积较小,形态均一;③培养第5天,K3表达量较多,p63表达量较少;培养第10天,K3和p63表达量均增多;培养第14天,K3表达量未见明显增多,p63表达量明显增多;④在无血清培养条件下,改良组织块培养法能显著促进角膜上皮干细胞的游离,提高体外培养细胞数量,为人角膜缘上皮组织片的构建提供种子细胞。ORCID: 0000-0001-8370-174X(许中中) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Establishment of an HIV cell-cell fusion assay by using two genetically modified HeLa cell lines and reporter gene

Infection of human cells with the human immunodeficiency virus type I (HIV-1) can be mimicked by a fusion process between cells expressing the HIV envelope protein (Env) and cells expressing both human CD4 together with the appropriate human chemokine receptors. In this study, a T-tropic HIV cell-cell fusion assay was established that utilized CD4, human CXCR4 and HIV NL4-3 gp160 as fusion components and a T7 polymerase-activated luciferase as a reporter system. The HeLa T4 cells used, expressed CD4 and CXCR4, and the applied HeLa KS386 cells expressed HIV NL4-3 gp160. By combining HeLa T4 cells with HeLa KS386 cells, an approximately about 100- to 300-fold increase in luciferase activity could be elicited relative to the control. The addition of anti-CD4 monoclonal antibody (Mab) (RPA-T4) or anti-CXCR4 Mab (12G5) in the assay significantly inhibited the fusion event; in contrast, an anti-CCR5 Mab (2D7) had no effect, indicating that the fusion assay was CD4 and CXCR4 dependent. In this report, fusion events could be monitored by both the luciferase reporter system and syncytia formation. Fusion events were monitored and compared using these two approaches. The luciferase reporter system was found to be more sensitive than syncytia formation. Moreover, compared with previous HIV fusion models, such as using recombinant vaccinia viruses, this system has several advantages, including simplicity and sensitivity. Finally, the system provides a powerful tool to study fusion mechanisms mediated by T-tropic HIV gp160, as well as to screen for fusion-blocking antibodies and antiviral agents.     

The biomedical potential of genetically modified flax seeds overexpressing the glucosyltransferase gene

Background

Flax (Linum usitatissimum) is a potential source of many bioactive components that can be found in its oil and fibers, but also in the seedcake, which is rich in antioxidants. To increase the levels of medically beneficial compounds, a genetically modified flax type (named GT) with an elevated level of phenylopropanoids and their glycoside derivatives was generated. In this study, we investigated the influence of GT seedcake extract preparations on human fibroblast proliferation and migration, and looked at the effect on a human skin model. Moreover, we verified its activity against bacteria of clinical relevance.

Methods

The GT flax used in this study is characterized by overexpression of the glucosyltransferase gene derived from Solanum sogarandinum. Five GT seedcake preparations were generated. Their composition was assessed using ultra pressure liquid chromatography and confirmed using the UPLC-QTOF method. For the in vitro evaluation, the influence of the GT seedcake preparations on normal human dermal fibroblast proliferation was assessed using the MTT test and the wound scratch assay. A human skin model was used to evaluate the potential for skin irritation. To assess the antimicrobial properties of GT preparations, the percentage of inhibition of bacterial growth was calculated.

Results

The GT seedcake extract had elevated levels of phenylopropanoid compounds in comparison to the control, non-transformed plants. Significant increases in the content of ferulic acid, p-coumaric acid and caffeic acid, and their glucoside derivatives, kaempferol, quercitin and secoisolariciresinol diglucoside (SDG) were observed in the seeds of the modified plants. The GT seedcake preparations were shown to promote the proliferation of normal human dermal fibroblasts and the migration of fibroblasts in the wound scratch assay. The superior effect of GT seedcake extract on fibroblast migration was observed after a 24-hour treatment. The skin irritation test indicated that GT seedcake preparations have no harmful effect on human skin. Moreover, GT seedcake preparations exhibited inhibitory properties toward two bacterial strains: Staphylococcus aureus and Escherichia coli.

Conclusions

We suggest that preparations derived from the new GT flax are an effective source of phenylopropanoids and that their glycoside derivatives and might be promising natural products with both healing and bacteriostatic effects. This flax-derived product is a good candidate for application in the repair and regeneration of human skin and might also be an alternative to antibiotic therapy for infected wounds.