Patent Application
20210115398
This application claims the benefits of the Taiwan Patent Application Serial Number 108137947, filed on Oct. 21, 2019, the subject matter of which is incorporated herein by reference.
The present invention relates to a corneal tissue culture system and a corneal tissue culture method, particularly, to a corneal tissue culture system and a corneal tissue culture method for increasing corneal endothelial cell density, healing of damaged corneal endothelial cell and stimulating corneal endothelial cell proliferation.
The lesion of the corneal tissue can be completely cured if the lesion is treated promptly and accurately in the early stage. But if the damage of the corneal tissue is too serious or the lesion of the corneal tissue is not treated in the early stage, the corneal transplantation must be performed to control the diseases. However, the number of the donor corneal tissue is rather low due to religious or traditional ideas. Accordingly, the number of corneal tissue donation is much lower than the demand for corneal tissue transplantation.
Please refer to
The endothelium layer of the corneal tissue is mainly composed of a monolayer of hexagonal flat endothelial cells, wherein the corneal endothelial cells are evenly and tightly arranged. However, the cell number of the endothelium layer will decrease with age and is not renewable. The defected area of the endothelium layer can only be filled by the expansion and migration of the adjacent cells. In contrast to corneal epithelial cells, human corneal endothelial cells are generally considered not expandable in vivo and slowly expandable in vitro. The endothelial cell density diminishes gradually from birth until death. The epithelium layer of the corneal tissue can be differentiated from limbal epithelial stein cells (LESC). Clinically, only donor corneas with an endothelial cell density greater than 2100 cells/mm2 are eligible because the endothelial cell density is a critical indicator for postoperative follow-up and assessments.
The endothelial cell density is a key factor for maintaining the physiological function of the corneal tissue; therefore, damage of the endothelium layer of the corneal tissue, low endothelial cell density, or endothelium layer dysfunction may cause the corneal edema and the donated corneal tissue difficult to manipulate during the surgery or even unusable. However, the conventional method to preserve the donor corneal tissue fails to focus on how to stimulate the proliferation of the corneal endothelial cells. For example, the conventional corneal preservation solution Optisol GS is disadvantageous of high cost, and the longer the storage time, the more cells will be lost.
In order to solve the aforementioned problems of lacking the donor corneal tissue, a technique for increasing the corneal endothelial cell density of the donor corneal tissue and restoring the endothelial cells of the declining corneal tissue is required to increase the availability of the donor corneal tissue to optimize the quality of the donor corneal tissue.
An object of the present invention is to provide a corneal tissue culture system, which is able to increase corneal endothelial cell density, heal damage corneal endothelial cells, and stimulate corneal endothelial cell proliferation.
Another object of the present invention is to provide a method of culturing a corneal tissue, wherein the corneal tissue is cultured in a specialized tissue culture dish allowing one portion of the corneal epithelium layer contact with air for simulating the corneal tissue in the real environment. The method of the present invention is effective in reducing the corneal edema; therefore, the quality of the donor corneal tissue may be improved.
The corneal tissue culture system of the present invention comprises a tissue culture media and a tissue culture dish, wherein the tissue culture media includes a basal medium and a lysophosphatidic acid (LPA), and the tissue culture dish accommodates the tissue culture media.
In one embodiment of the present corneal tissue culture system, the concentration of the lysophosphatidic acid in the tissue culture media is 10 μmol/L to 25 μmol/L, wherein 20 μmol/L is preferable.
In one embodiment of the present corneal tissue culture system, the basal medium is Dulbecco's modified Eagle's medium (DMED).
In one embodiment of the present corneal tissue culture system, the tissue culture media further includes a fetal bovine serum (FBS), wherein the weight percentage of FBS in the tissue culture media is 5%-20%, wherein 10% is preferable.
In one embodiment of the present corneal tissue culture system, the tissue culture media further includes an antibiotic agent, the antibiotic agent can be selected from those antibiotic agents known in the art. For example, the antibiotic agent can be at least one selected from the group consisting of gentamicin, penicillin, streptomycin, and mixture thereof, wherein gentamicin is preferable.
In one embodiment of the present corneal tissue culture system, the concentration of the antibiotic agent is 20-30 μg/mL, wherein 25 μg/mL of gentamicin is preferable.
In the present corneal tissue culture system, LPA is purchased from Sigma-Aldrich; DMEM, FBS, and gentamicin are purchased from Invitrogen.
In one embodiment of the present corneal tissue culture system, the tissue culture dish is a petri dish, wherein the petri dish is selected from any types of petri dish known in the art without limitation as long as the corneal tissue is able to be immersed in the tissue culture media.
In one embodiment of the present corneal tissue culture system, the tissue culture dish includes: a dish body having a first accommodating space and a first opening; and a tube body disposed in the first accommodating space, and having a second accommodating space and a second opening, wherein the second opening and the first opening face toward a first direction; wherein the tissue culture media is filled in the first accommodating space and the second accommodating space. In one embodiment, the tube body may be obtained by cutting down a portion of the 1000 μL micropipette. For example, 10 mm of the tube body may be cut from the 1000 μL micropipette and fixed at the center of the petri dish.
In one embodiment of the present corneal tissue culture system, a height of the tube body is smaller than a height of the dish body.
The method of culturing corneal tissue includes the steps of: (1) providing a tissue culture dish having a dish body and a tube body, wherein the dish body has a first accommodating space and a first opening, the tube body is disposed in the first accommodating space and has a second accommodating space and a second opening, the first opening and the second opening face toward a first direction; (2) filling a tissue culture media in the first accommodating space and the second accommodating space; and (3) disposing a corneal tissue on the second opening, wherein the corneal tissue has an epithelial cell layer facing the first direction, wherein at least a portion of the corneal tissue contacts with the tissue culture media.
In detail, please refer to the cross-sectional view of the corneal tissue illustrated in
According to one embodiment of the present method of culturing corneal tissue, in step (3), the at least a portion of the corneal tissue is at least a portion of the epithelial cell layer.
According to one embodiment of the present method of culturing corneal tissue, in step (3), the at least a portion of the corneal tissue is the corneal tissue as a whole, and the corneal tissue is immersed in the tissue culture media without contacting the air.
According to one embodiment of the present method of culturing corneal tissue, in step (1), a height of the tube body is smaller than a height of the dish body.
According to one embodiment of the present method of culturing corneal tissue, in step (2), the tissue culture media includes a basal medium and a lysophosphatidic acid, wherein the concentration of the lysophosphatidic acid in the tissue culture media is 10 μmol/L-25 μmol/L, wherein 20 μmol/L is preferable.
According to one embodiment of the present method of culturing corneal tissue, in step (2), the basal medium is DMEM.
According to one embodiment of the present method of culturing corneal tissue, in step (2), the tissue culture media further includes a fetal bovine serum, wherein the weight percentage of FBS in the tissue culture media is 5%-20%, wherein 10% is preferable.
According to one embodiment of the present method of culturing corneal tissue, the tissue culture media further includes an antibiotic agent, which is at least one selected from the group consisting of gentamicin, penicillin, streptomycin, and mixture thereof, wherein gentamicin is preferable.
According to one embodiment of the present method of culturing corneal tissue, in step (2), the concentration of the antibiotic agent is 20-30 μg/mL, wherein 25 μg/mL is preferable.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
[Statistical Analysis]
All numeric results are shown as the mean±standard deviation. The data were compared using one-way ANOVA with Microsoft Excel version 2016 (Microsoft, Redmont, Wash.). The data were analyzed using two-tailed p values, where P<0.05* and P<0.01** were considered to indicate significant differences.
[Corneal Tissue Culture System]
Please refer to the corneal tissue culture system 1000 of the first embodiment illustrated in
Please refer to the corneal tissue culture system 2000 of the second embodiment of the illustrated in
[The Observation of the Cultured Corneal Tissue]
Firstly, a rabbit corneal tissue was placed in the tissue culture dish 100, cultivated with the tissue culture media 300 consisting of DMEM basal medium, 10% FBS, and 25 μg/mL gentamycin, and incubated at 37° C. with 5% CO2, wherein a portion of the epithelium layer of the rabbit corneal tissue contacts with air (The tissue culture system 1000 as illustrated in
[Dose-Response Test]
The tissue culture media (DMEM, 10% FBS, 25 μg/mL gentamycin) added with 0 μmol/L, 10 μmol/L, 20 μmol/L, and 100 μmol/L of LPA were used for co-cultivating the rabbit corneal endothelial cells and the rabbit corneal stroma cells at 37° C. with 5% CO2. After 5 days of cultivation, the cell number of the rabbit corneal endothelial cells of each groups were calculated and shown in
[Evaluation of the Corneal Endothelial Cell Density of the Corneal Tissue]
The rabbit corneal tissues were placed individually in the tissue culture dishes 100, cultivated with the tissue culture media (DMEM basal medium, 10% FBS, and 25 μg/mL gentamycin) added with 20 μmol/L LPA (Example) or without LPA (Comparative example), and incubated at 37° C. with 5% CO2, wherein a portion of the corneal epithelium layer of the rabbit corneal tissues faced toward the first direction A and contacted with air and the corneal endothelium layer contacted with the tissue culture media. After 7 days of cultivation, the cell morphology and the analysis of cell density of the corneal endothelium layer were shown in
[Morphology of the Corneal Endothelial Cells of the Corneal Tissue]
The rabbit corneal tissues were placed individually in the tissue culture dishes 100, cultivated with the tissue culture media (DMEM basal medium, 10% FBS, and 25 μg/mL gentamycin) added with 10 μmol/L LPA (Example) or without LPA (Comparative example), and incubated at 37° C. with 5% CO2 for 7 days, wherein a portion of the corneal epithelium layer of the rabbit corneal tissues faced toward the first direction A and contacted with air and the corneal endothelium layer contacted with the tissue culture media. The corneal endothelium layer was immunostained for ZO-1 on day 0 and day 7 for observing the cell morphology of the corneal endothelial cells of the corneal tissue. The results shown in
[Evaluation of the Cell Proliferation of the Corneal Tissue]
The rabbit corneal tissues were placed individually in the tissue culture dishes 100, cultivated with the tissue culture media (DMEM basal medium, 10% FBS, and 25 μg/mL gentamycin) added with 10 μmol/L LPA (Example) or without LPA (Comparative example), and incubated at 37° C. with 5% CO2 for 7 days, wherein a portion of the corneal epithelium layer of the rabbit corneal tissues faced toward the first direction A and contacted with air and the corneal endothelium layer contacted with the tissue culture media. Bromodeoxyuridine (BrdU) was added to the culture media from day 5 to day 7 for observing the proliferation of the cells. The BrdU labeling cells (green), shown in
[Investigation of the Healing Effect of the Damaged Corneal Endothelium Layer of the Corneal Tissue]
A polyvinylidene fluoride (PVDF) filter membrane with regularly punched-out holes was used to cover the rabbit corneal endothelium layer; exposing the inner damage zone (I) scraped with Cellulose Eye Spears. The rest area of the corneal endothelium layer was defined as the outer damage zone (O). The damaged corneal tissues were placed individually in the tissue culture dishes 100, cultivated with the tissue culture media (DMEM basal medium, 10% FBS, and 25 μg/mL gentamycin) added with 20 μmol/L LPA (Example) or without LPA (Comparative example), and incubated at 37° C. with 5% CO2, wherein a portion of the corneal epithelium layer of the rabbit corneal tissues faced toward the first direction A and contacted with air and the corneal endothelium layer contacted with the tissue culture media. The inner damage zone (I) and the outer damage zone (O) of the corneal tissues on day 0 and day 2 were observed using Alizarin Red S staining. The morphology of the corneal tissue stained by Alizarin Red S was shown in
[Evaluation of Corneal Tissue Transplantation]
The damaged corneal tissues were prepared using the PVDF filter membrane described above (please refer to the above paragraph). Then, those damaged corneal tissues were placed individually in the tissue culture dishes 100, cultivated with the tissue culture media (DMEM basal medium, 10% FBS, and 25 μg/mL gentamycin) added with 20 μmol/L LPA (Example) or without LPA (Comparative example 1), and incubated at 37° C. with 5% CO2, wherein a portion of the corneal epithelium layer of the rabbit corneal tissues faced toward the first direction A and contacted with air and the corneal endothelium layer contacted with the tissue culture media. After 2 days of cultivation, the damaged corneal tissues were transplanted into rabbit eyes. In addition, total corneal endothelium scrapping and undamaged mock donor corneas were also prepared to serve as comparative examples 2 and 3. External eye photography was taken in the rabbits after transplantation on day 0 and day 6, respectively. Following sacrifice on day 6, the corneal endothelial cells were stained with Alizarin Red S for observations of the cell morphology of the corneal endothelial cells in all density zones (A) (average of high-density and low-density zones) and cell shape in low-density zone (L) were examined. The results were shown in
[Gene Expression Analysis Using DNA Microarray]
The gene expression profiling and the cytokine of the human corneal stromal cells were analyzed using cDNA microarray chip and Cytokine Array G5 chip. The human corneal stromal cells were cultured with culture media (DMEM basal medium, 10% FBS, and 25 μg/mL gentamycin) added with 20 μmol/L LPA (served as an Example) or without adding LPA (Comparative example) and incubated at 37° C. with 5% CO2. After 2 days of cultivation, the total RNA from the human corneal stromal cells in example and comparative example were isolated with the TRIzol reagent and purified using RNeasy column. After amplification and labeling the total RNA samples, GeneChip hybridization was performed and a GeneArray G7 scanner was used to scan the results. The culture media from the example and the comparative example were collected and the cytokines were analyzed using a human cytokine antibody array. The results were shown in Table 1, wherein CCL20 and IL-1β (IL1B) gene expressions were significantly up-regulated out of 16 genes with high expressions in the LPA-treated example. Refer to Table 2., CCL20 and IL-1β (IL1B) cytokines also showed higher secretion in the LPA-treated example. Accordingly, the secretion of CCL20 and IL-1β were significantly increased when treated with LPA. It was confirmed that LPA stimulated the proliferation of the endothelial cells of the corneal tissue via two pathways including directly affecting the endothelial cells and stimulating the secretion of CCL20 and IL-1β from the endothelial cells.
[Evaluation of the Proliferation and Morphology of the Corneal Endothelial Cells and of the Corneal Tissue]
The cell morphology, cell density, and cell proliferation of the present evaluation were performed through immunostaining.
One-third of the human corneal residual tissue samples were used for immunostaining for ZO-1 directly (Day 0), while the remaining two-thirds were immersed and placed endothelial side up in the tissue culture media (DMEM basal medium, 10% FBS, and 25 μg/mL gentamycin) added with 20 μmol/L LPA (served as an Example) or without adding LPA (Comparative example) and incubated at 37° C. with 5% CO2. 7 days later, the tissues were cut into halves and subjected to immunostaining for ZO-1 and Ki-67 to evaluate the effects of LPA treatment on corneal endothelial cell density and cell proliferation. As shown in
Base on the aforementioned experimental results, the corneal tissue culture system of the present invention is advantageous for increasing corneal endothelial cell density, healing of damaged corneal endothelial cell, and stimulating corneal endothelial cell proliferation by adding LPA.
| Number | Date | Country | Kind |
|---|---|---|---|
| 108137947 | Oct 2019 | TW | national |
