RU2809076C1 - Method of obtaining limbal stem cells in collagen hydrogel - Google Patents

Abstract

FIELD: medicine; biotechnology; ophthalmology.

SUBSTANCE: invention can be used to obtain limbal stem cells. A limbal tissue biopsy is isolated with a section of the sclera, fermented with a 2% dispase solution at 37°C for 1 hour. Fragmented and fermented again using 0.25% trypsin solution in EDTA at 37°C for 20 min. The resulting cell suspension is cultured under conditions of 5% CO₂ at 37°C within 14 days. 1 ml of suspension containing 0.5×10⁶ cells are placed in a mixture consisting of 1 ml of Viscoll PA 8 collagen solution, 1 ml of neutralizing buffer solution, then resuspended. The resulting hydrogel with cells in its composition is cultivated under conditions of 5% CO₂ at 37°C for 7 days.

EFFECT: invention ensures obtaining of cultured cells with maximally preserved characteristics and prolonged biodegradation, which creates prerequisites for their use in the treatment of limbal insufficiency.

1 cl, 1 ex

Description

The invention relates to biotechnology and ophthalmology, and can be used to obtain limbal stem cells in collagen hydrogel.

The cornea is the main component of the optical media of the eye and has 4/5 of the refractive power of the eye. The corneal epithelium is multilayered, non-keratinizing and is in constant renewal in a physiological state and in response to damage, this is provided by both limbal stem cells (LSCs) of the corneal part of the limbus in the Vogt niche, and mesenchymal stem cells of the limbus. When LSC are damaged or their microenvironment (limbal niche) is disrupted under the influence of chemical, mechanical, autoimmune and other factors, limbal insufficiency (LI) develops. This is accompanied by neovascularization and loss of transparency of the cornea, disruption of its epithelial layer, pathological growth of the conjunctiva and, as a consequence, decreased visual acuity (Deng S.X., Borderie V., Chan S.S., et al. Global consensus on definition, classification, diagnosis, and staging of limbal stem cell deficiency // Cornea. 2019. Vol. 38, N 3. P. 364-375. doi: 10.1097/ICO.0000000000001820, Deng S.X., Kruse F., Gomes J.A.P., et al. Global consensus on the management of limbal stem cell deficiency. Cornea. 2020;39: 1291-1302, Ramachandran C., Basu S., Sangwan V.S., et al. Concise review: the coming of age of stem cell treatment for corneal surface damage // Stem Cells Transl Med. 2014. Vol. 3, N 10. P. 1160-1168. doi: 10.5966/sctm.2014-0064). Treatment of LN includes a set of measures aimed at regenerating the epithelium and restoring the function of the limbal niche. At the moment, there are both therapeutic and surgical approaches to solve this problem.

Conservative methods involve symptomatic treatment with the help of various drugs (tear substitutes, anti-inflammatory drugs), therapeutic contact lenses, as well as autologous blood derivatives, which contain various growth factors that stimulate corneal epithelization and activate pro-inflammatory cytokines. The disadvantages of conservative treatment include the lack of targeted effects on the affected tissue and the ability to provide a long-term therapeutic effect. In addition, the need for frequent installations leads to a decrease in the patient’s quality of life. In this regard, the current emphasis in the treatment of LN is shifting to surgical techniques, including limbal transplantation and cultured cell transplantation.

There is a known method for obtaining a transplant for the treatment of limbal insufficiency (invention patent RU 2720470 dated April 30, 2020), including mechanical cleaning of the donor sclera, double sequential treatment with hydrogen peroxide, ammonium hydroxide, soaking, freezing, heating, fragmentation, cultivation on their concentrated cell suspension of multipatent mesenchymal stem cells and progenitor cells for use in the treatment of LN.

The disadvantage of this method is that this graft is a decellularized fragment of the donor allogeneic sclera, and the donor tissue retains some of the antigens and can cause a reaction of the recipient's body to the graft.

There is a known method for producing limbal cells for the treatment of limbal insufficiency using a tissue-engineered construct consisting of cultured LSCs grown on a feeder layer of fibroblasts and decellularized amniotic membrane (invention patent CN 1635115 dated 07/06/2005). The disadvantage of this method is the relative difficulty in preparing the graft. In addition, the sources for obtaining fibroblasts and LSCs are fibroblasts from the limbal stroma of newborns, a fresh donor eye, the contralateral healthy eye of the patient or the eyes of relatives, and there is a high risk of transmission of infection when using the amniotic membrane.

There is a known method for obtaining a graft for the treatment of LN (Regeneration of corneal epithelium utilizing a collagen vitrigel membrane in rabbit models for corneal stromal wound and limbal stem cell deficiency. / J.J. Chae, W.M. Ambrose, F.A. Espinoza et al. // Acta Ophthalmologica. - 2015; 93(l):e57-e66). using Vitrigel collagen, which contains a low concentration of collagen - 0.5% - as a carrier for culturing LSCs in the treatment of pre-created total LN caused by a chemical burn.

The disadvantage of this method is that, due to the low concentration of collagen, the carrier has unsatisfactory biomechanical properties, which leads to difficulties during surgery when performing surgical manipulations. In this regard, for fixation, sutures to the sclera and additional fibrin glue were used, which is a commercial, inaccessible material and has low adhesive-cohesive strength and relatively low mechanical properties.

The closest analogue of the proposed method is a method of the same purpose with the cultivation of LSCs based on a collagen carrier (Bezushko A.V., Dubovikov A.S., Kulikov A.N., Churashov S.V., Chernysh V.F., Blinova M. I., Aleksandrova O.I., Khorolskaya Yu.I., Gavrilyuk I.O., Karpovich V.V., Danilichev V.F. Application of a collagen scaffold and amniotic membrane with cultured limbal stem cells to eliminate limbal insufficiency: an experimental study . // Pacific Medical Journal. - 2019. - No. 2. P. 54-57). The disadvantage of this method is the use of gelatin made from type I collagen with a low concentration (2 mg/ml), which quickly biodegrades and is not able to maintain an optimal environment for cultured LSCs for a long time. In addition, due to the high water content, this carrier has lower biomechanical properties.

The objective of the invention is to further develop methods for obtaining LSCs based on collagen hydrogels.

The technical result of the proposed method is the production of cultured cells with the most preserved characteristics and prolonged biodegradation.

The technical result is achieved by obtaining limbal cells by two-stage fermentation with dispase and trypsin and cultivation on a biopolymer carrier based on type I collagen Viscoll RA 8. Due to the peculiarity of the structure of the limbus with a limbal niche immersed deep during collection, a biopsy of the limbal tissue is isolated with a section of the sclera, From the stroma of the limbal tissue, limbal mesenchymal stem cells are then isolated during cultivation, which regulate the functions of LSCs and will maintain cellular homeostasis in the limbus after transplantation in the treatment of limbal insufficiency.

Double fermentation allows you to quickly obtain a large volume of cell mass: in the first stage, with the help of dispase, the entire epithelium is separated from the stroma along the basement membrane. The second fermentation uses trypsin to separate the tissue into individual cells in suspension.

A hydrogel with an increased concentration of native, chemically unmodified collagen Viscoll PA 8 (http://imtek.ru/catalog/products/cell-culture/viscoll/Viscoll_Kit_for_3D_bioprinting_and_cell_culture/) allows one to reproduce an architecture close to the natural niche of limbal stem cells. In this case, the cells are not on the surface, but are distributed in the structure of the hydrogel.

The combination of techniques makes it possible to obtain LC with maximum yield, survival, and preservation of the properties of cultured cells. Using the proposed method, limbal cells in a hydrogel were obtained from 5 male chinchilla rabbits with an initial body weight of 2.5-3.0 kg.

Staining with vital dyes (Calcein AM and Heoest) and immunofluorescence analysis were performed to assess the characteristics of cultured cells, including viability, proliferative activity and cell phenotype. At all stages of cultivation, the morphology of cells was assessed; phase-contrast microscopy was used for observations.

On the 1st day, the cells began to adhere to the bottom of the Petri dish. On the 10th day of cultivation, a loose monolayer was formed, and by the 14th day, a dense subconfluent layer was formed. On the 14th day, the cultured cells were removed from the surface of the culture flask, and after counting, it was revealed that by this time the culture already had 0.5 × 10 ⁶ cells per 1 ml.

According to the results of vital staining with Calcein-AM, it was shown that the survival of cells on the membrane was high and amounted to 92.0±2.6%. Immunofluorescent staining showed that the culture contained a high concentration of proliferating cells staining positively for the Ki67 protein. When assessing the phenotype of the resulting cell cultures, it was revealed that most cells were stained with K3/12 or Vimentin. About 20% of the cells were positive for the P63 marker and formed characteristic round colonies, which indicated the presence of a population of LSCs in the cells obtained from the biopsy specimen. The immunofluorescent phenotype of the cells also remained intact when cultured in a hydrogel collagen construct. In addition, a relatively high proliferation of cultured cells was revealed. The limbal cells obtained by the proposed method in the composition of a collagen hydrogel can be used for the treatment of LN, as well as as a source of LSC for the restoration of any defects in the corneal epithelium by transplantation into the damaged area.

The method is carried out as follows.

A limbal tissue biopsy is isolated with a section of the sclera and fermented with a 2% dispase solution at 37°C for 1 hour. Fragment and ferment again using 0.25% trypsin in EDTA at 37°C for 20 minutes. The resulting cell suspension is cultured under conditions of 5% CO ₂ at 37°C for 14 days. 1 ml of suspension containing 0.5×10 ⁶ cells is placed in a mixture consisting of 1 ml of Viscoll PA 8 collagen solution, 1 ml of neutralizing buffer solution, then resuspended. The resulting hydrogel with cells in its composition is cultured under conditions of 5% CO ₂ at 37°C for 7 days.

Example.

A male chinchilla rabbit No. 1, weighing 3.5 kg, had his left eye operated on. Subconjunctival anesthesia was performed with 2% lidocaine - 0.5 ml. An incision was made along the limbus for 2 hours. The conjunctiva and Tennon's membrane were separated and hemostasis was performed. A tissue fragment measuring 2 × 4 mm was cut out using a sharp-pointed ophthalmic blade, retreating 1 mm from the side of the cornea and conjunctiva to a depth of 200 μm. The operation was completed with the application of two interrupted sutures and the injection of 0.3 ml of 0.4% dexamethasone.

The removed tissue was washed, as is customary, with Hanks' solution containing antibiotics (gentamicin 0.16 mg/ml and streptomycin 1 mg/ml) and amphotericin B (5 mg/ml). The biopsy was then placed in a 2% dispase solution in DMEM and fermented for 1 hour at 37°C. Fragmentation was then carried out and washed with EDTA solution, followed by secondary fermentation with a solution of 0.25% trypsin in EDTA for 20 minutes. at 37°C. Trypsin was inactivated with DMEM/F12 medium containing serum (10% FBS-Fetal Bovine Serum), after which limbal fragments were pipetted, followed by centrifugation at 400 g (rpm) for 6 min. The supernatant was discarded, the residue with undissolved pieces of the limbus and individual cell conglomerates was resuspended, as is customary, in the DMEM/F12 nutrient medium with the addition of 10% fetal calf serum, 2 mM glutamine, 10 ng/ml rEGF, 5 ml of the ready-made reagent Insulin-Transferrin- Selenium, 10000 U/mL penicillin/streptomycin. Cells were cultured under 5% CO2 at 37°C for 14 days. The medium was changed every two days. Cells were counted using a TC20 automatic cell counter. On day 14, the cultured cells were removed from the surface of the culture flask. 1 ml of collagen solution (Viscoll PA 8) was thoroughly mixed with 1 ml of neutralizing buffer solution (Tris 80 mM, H Fne 220 μg/ml, DMEM) and 1 ml of cell suspension with a concentration of 0.5×10 ⁶ cells per 1 ml was added to the resulting mixture , the final mixture was stirred until homogeneous. Then 2 ml were poured onto a sterile Petri dish d=90 mm, additionally squeezing with a sterile Petri dish d=35 mm with light movements to even out the thickness of the hydrogel disk. After the formation of disks from the hydrogel with LSC, 10-15 ml of nutrient medium was added to the dishes and further cultivated for 7 days. The obtained LSCs in the hydrogel composition were studied to evaluate their characteristics.

Thus, the proposed method makes it possible to obtain LSCs with preserved properties, which creates prerequisites for their use in the treatment of limbal insufficiency.

Claims (1)

A method for obtaining limbal stem cells, including obtaining a limbal tissue biopsy, fermentation, fragmentation and cultivation, characterized in that the limbal tissue biopsy is isolated with a section of the sclera, fermented with a 2% dispase solution at 37°C for 1 hour, after fragmentation it is fermented again using a 0.25% solution of trypsin in EDTA at 37°C for 20 minutes, the resulting cell suspension is cultured under 5% CO ₂ at 37°C for 14 days, then 1 ml of a suspension containing 0.5×10 ⁶ cells, placed in a mixture consisting of 1 ml of Viscoll PA 8 collagen solution, 1 ml of neutralizing buffer solution, then resuspended and the resulting hydrogel with cells in its composition is further cultivated under conditions of 5% CO ₂ at 37°C for 7 days.