Method for separation and organotypic preservation of allogenic limb transplant

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely ophthalmology, and may be used in separation and organotypic preservation of an allogenic limb transplant. A method involves early surgical separation of the limb transplant from a whole corpse donor eyeball with eliminated perforation after donor's death. The transplant is preserved within 28 days in a medium containing insulin, dexamethasone, blood serum, penicillin, streptomycin, dextran 40000, HEPES M-solution, 7.5% bocarbonate, 40% glucose, L-glutamine, penicillin, streptomycin, amphotericin B and Hanks salt medium 199 under certain proportion of the components. The preservation is performed at temperature 37°C with the medium changed every 3 days. The same medium, but serum-free is used for washing. The method provides preserving transplant viability with proliferating progenitor cells that leads to achieving stable, long-term epithelisation, stroma defect recovery, prevention of corneal conjunstivisation.

EFFECT: prepared transplant may be used for surgical management of the patients with limb cell insufficiency syndrome of various aetiologies.

2 cl, 1 ex, 9 dwg

 

The invention relates to ophthalmology, and can be used in the surgical treatment of patients with the syndrome of limbus cell failure (LCN) of various etiologies.

To date, all proposed methods of treatment LCN are divided into two main groups.

1. Cytotherapy cultured limbus epithelial cells on different carriers. As the carrier can speak: amniotic membrane [Grueterich m, Scheffer C., Tseng G. Human Limbal Progenitor Cells Expanded on Intact Amniotic Membrane Ex Vivo // Arch Ophthalmol. 2002; 120:783-790; Koizumi N., Cooper, L.J., N.J. Fullwood et al. An evaluation of cultivated corneal limbal epithelial cells, using cell-suspension culture Invest. Ophthalmol. Vis. Sci. 2002. 43(7):2114-21], fibrin substrate [Sudha Century, H.N. Madhavan, Sitalakshmi g et al. Cultivation of human corneal limbal stem cells in Mebiol gel is A thermo-reversible gelation polymer // Indian. J. Med. Res. 2006. Dec; 124(6):655-64] and a soft hydrogel contact lens [Sangwan V.S. Limbal stem cells in health and disease // BioSci. Rep. 2001. Vol.21. P.385-405].

The disadvantage of this method is the technical complexity and high cost of implementation, and the need to use feeder layer under cultivation.

2. Limbus transplantation [Kenyon K.R., S.C. Tseng // Ophthalmology. - 1989. - Vol.96. N5. - P.709-722].

The known method autotransplantation limbus flap, taken with a healthy bilateral eye recipient [patent No. 2348384 from 10.03.2009].

The disadvantage of this method is the inability excision quite a large shopping Mall is the area of the limb, and the disagreement of many patients for this procedure.

The known method of allogeneic transplantation "fresh" limbus flaps isolated from cadaveric donor eyes received in ophthalmic institutions transplanting the cornea [Dua HS, Blanco AA. Allolimbal transplantation in patients with limbal stem cell defi ciency // Br J Opthalmol., 1999. 83: 414-19; RF Patent №2361550 from 20.07.2009].

The disadvantage of this method is high risk of transplant rejection due to the presence of a large number of Langerhans cells expressing HLA-DR antigens, which creates the need for immunosuppressive therapy.

The closest analogue is the allocation method and organo-typical cultivation limbus grafts described in the publication Zito-Abbad E, Borderie VM, Baudrimont M, et al. Corneal epithelial cultures generated from organ-cultured limbal tissue: factors influencing epithelial cell growth // Curr Eye Res. 2006 May; 31(5): 391-9.

How is that organo-typical cultivation are corneoscleral ring remaining after removal of corneal transplant for end-to-end or layer-by-layer keratoplasty, including: processing of 0.05%trypsin solution for 1 hour and disposal II (1,2 U/ml) for 15 minutes at 37°C, further cultivation using a feeder layer of human keratocytes in DMEM/F12 (Dulbecco''s Modified Eagle's Medium Nutrient Mixture F-12 US with the addition of fetal bovine serum, insulin, dexamethasone, cholera toxin, gentamicin within 28 days in CO2-incubator at a temperature of 31°C, with 5%content of CO2and 99% humidity, replacing medium every 3 days. The advantage of this method is the reduction of the expression of HLA-DR antigens by natural elimination of Langerhans cells from the limbus of the graft due to long-term (28 days) organo-typical cultivation [Holland E.J, DeRuyter D.N., Doughman D.J. Langerhans cells in organ-cultured corneas // Arch Ophthalmol 1987; 105: 542-5].

However, the described method has several disadvantages:

for organo-typical cultivation used donor corneoscleral ring, after removal of corneal transplant, in this regard, the length of time from the death of the donor before cutting out the limbus grafts is not less than 72 hours, which adversely affects survival and proliferative activity of limbus epithelial stem cells;

- the necessity of using special tools for cutting out the limbus allografts, as well as the inability to form the limbus transplant uniform thickness and the necessary forms with knives-rasseivatelei of corneoscleral rings;

- used during processing corneoscleral what about the ring of the high concentration of enzymes (trypsin and dispose II) leads to a large loss of limbus epithelial cells (Figure 1, 2).

- cultivation at 31°C promotes proliferation only epithelial cells of the limb, which leads to the necessity to use under cultivation feeder layer (Figure 3).

The task of the invention is to develop a method of preoperative preparation of the surgical selection and organo-typical conservation) allogeneic limbus transplant for transplant patients with LCN.

The technical result is to achieve stable, long-epithelialization, the restoration of defects stroma and prevention of conjunctivitis of the cornea at the limbus cell failure.

The technical result is achieved in that in the method of preoperative preparation of allogeneic limbus transplant for transplant patients with limbus cell failure, including surgical selection limbus grafts from cadaveric donor eyes, organo-typical conservation within 28 days in medium containing insulin, dexamethasone, serum, antibiotics, replacing the medium every 3 days, laundering, according to the invention the surgical selection of limbus transplants performed from a single cadaveric donor of the eyeball, except for its perforation, organo-typical conservation is carried out at a temperature of 37°C, and the environment for organo-typical conse the activate your product further comprises dextran 40000, HEPES odnokolernyh, a 7.5%solution of bicarbonate, 40%solution of glucose, L-glutamine, amphotericin b and medium 199 on the Hanks salts, and contains antibiotic penicillin and streptomycin in the following ratio of the components in 1000 ml solution:

dextran 4000050.0 g
HEPES odnokolernyh15.0 ml
a 7.5%solution of bicarbonate10.0 ml
40%glucose solution5.0 ml
L-glutamine1.5 g
penicillin250000 units
streptomycin200.0 mg
amphotericin b1.4 mg
insulin "Humulin of regular"0.8 ml
dexamethasone with both molarity 10-81.0 ml
serum human blood
or cattle100,0 ml
medium 199 salts Hanksrest

while laundering from the whey produced the claimed medium not containing serum.

As serum can be used fetal bovine serum or human serum, cord blood or human serum IV blood group.

Catalog number medium 199 salts Hanks - S230 LLC Paneco".

The advantage of this method is the possibility of taking limbus of the graft in the early stages after the death of the donor, comprising not more than 18 hours, which contributes to maintaining reserves the viability of the cells (Figure 4).

The advantage of this method is the technical simplicity of cutting out the limbus of the graft with the entire circumference of the limb whole cadaveric donor eyes, when this is achieved a uniform thickness graft, not more than ½ of the thickness of the sclera, which prevents perforation of the donor's eyeball, to perform subsequent dissection of corneoscleral disc, preservation and planned through or layer-by-layer keratoplasty.

The proposed method organo-typical conservation limbus grafts can preserve the natural microenvironment of limbus epithelial progenitor cells, namely, mesenchymal stem cells, which are goods which owing to its paracrine properties fulfil the function of a feeder layer.

The preservation of the structure of limbus graft with viable, proliferating progenitor cells in the process of implementation of the method makes it possible to further cryopreservation limbus grafts and long-term storage in liquid nitrogen at a temperature of -196° to create a Bank of limbus grafts (Figure 5, 6).

The method is as follows. From cadaveric eyeball prepared for the donor cornea on medical technology "Algorithm procurement of cadaveric donor human corneas for transplantation" (of IRTC "eye microsurgery" named. Acad. Fyodorov") in sterile ward, after decapitalization of the cornea and remove the bulbar conjunctiva with the entire surface of the diamond or metal blade are 2 circular cut: one - sclera, retreating to 2 mm from the transparent part of the cornea to a depth of 0.5 mm; the other on the boundary between the limbus and transparent areas of the cornea. Next on the same depth, perpendicular incision is made in the direction of the cornea are 4 holes in the 3, 6, 9 and 12 hours, then a knife-rasseivatele tsaparevetsa tissue layer at a given depth, holding the resulting flap of corneal forceps, the result of limbus grafts stretch fabric with a width of 2 mm and thickness is Noah 0.5 mm over the entire length (Figure 7, 8, 9).

Received limbus grafts are placed in a sterile vial of 25 cm2and filled with 4 ml of medium for organo-typical conservation, which can be obtained by mixing the components according to the formula of the invention or may be obtained from the Solution for the storage of the cornea" (THE No. 9398-013-29039336-2008 produced LLC NEP "eye microsurgery", Moscow, Beskudnikovsky Boulevard, building 59A), including dextran, HEPES, bicarbonate, glucose, L-glutamine, penicillin, streptomycin, amphotericin b and medium 199 on the Hanks salts, which is optionally added insulin, dexamethasone and serum of humans or cattle, when the ratio of the components in 1000 ml solution: dextran 40000 - 50.0 g, HEPES odnokolernyh - 15.0 ml of 7.5%solution of bicarbonate - 10.0 ml, 40%glucose solution to 5.0 ml, L-glutamine and 1.5 g, penicillin - 250000 units, streptomycin - 200.0 mg, amphotericin b - 1.4 mg, insulin "Humulin of regular" - 0.8 ml, dexamethasone with both molarity 10-8to 1.0 ml serum human or cattle in 100.0 ml medium 199 salts Hanks - the rest.

Conservation is carried out for 28 days at 37°C by incubation in an atmosphere of air with 5% CO2with 99% humidity. Replacement conservation of the environment is provided every 3 days. Before using limbus transplant washed in the environment such as the level, but free from serum, namely when the ratio of the components in 1000 ml: dextran 40000 - 50.0 g, HEPES odnokolernyh - 15.0 ml of 7.5%solution of bicarbonate - 10.0 ml, 40%glucose solution to 5.0 ml, L-glutamine and 1.5 g, penicillin - 250000 units, streptomycin - 200.0 mg, amphotericin b - 1.4 mg, insulin "Humulin of regular" - 0.8 ml, dexamethasone with both molarity 10-8to 1.0 ml medium 199 salts Hanks - the rest.

Then limbus graft is transferred into a sterile tube with the medium of the same composition, in which laundered, that is free of serum and in the cool bag at a temperature of 4-6°C is delivered to the operating room.

Example. Patient K., 54 years old.

Diagnosis: Chronic recurrent erosion of cornea of both eyes.

Anamnesis morbi: suffering for 5 years, receive conservative therapy.

Anamnesis vitae: 6 years ago was held radiation and chemotherapy on breast cancer.

Status localis: visual acuity in both eyes (OU) of 0.1 not corrects.

OU - photophobia, in the center of the cornea decapitalization.

Under conjunctival limbus in the upper segment of the right eye produced transplant was prepared according to the proposed method limbus transplant, at the same time as the serum used human serum IV blood group.

The operation and the postoperative period was uneventful, was called ACANA standard local immunosuppressive therapy. After surgery on the 7th day was marked complete epithelialization of the cornea. A month later conducted a similar operation on my left eye. When control inspections throughout the entire observation period (6 months) signs of rejection limbus grafts were observed, cornea epithelisation completely transparent, visual acuity in both eyes of 0.6-corrected sph+1,0D=0,9.

Thus, the proposed method of treatment (surgical selection and organo-typical conservation) allogeneic limbus grafts allows to achieve stable and long-epithelialization of the cornea in patients with limbus cell failure.

1. Method of preoperative preparation of allogeneic limbus transplant for transplant patients with limbus cell failure, including surgical selection limbus grafts from cadaveric donor eyes, organo-typical conservation within 28 days in medium containing insulin, dexamethasone, serum, antibiotic, replacing the medium every 3 days, laundering, characterized in that the surgical selection of limbus transplants performed early after the death of the donor, from a single cadaveric donor of the eyeball, except for its perforation, organo-typical conservation is carried out at a temperature of 37°C, and the environment for organo-is epicheskoi conservation further comprises dextran 40000, HEPES odnokolernyh, a 7.5%solution of bicarbonate, 40%solution of glucose, L-glutamine, amphotericin b, medium 199 on the Hanks salts, and contains antibiotic penicillin and streptomycin in the following ratio of the components in 1000 ml solution:

dextran 4000050.0 g
HEPES odnokolernyh15.0 ml
a 7.5%solution of bicarbonate10.0 ml
40%glucose solution5.0 ml
L-glutamine1.5 g
penicillin250000 units
streptomycin200.0 mg
amphotericin b1.4 mg
insulin "Humulin of regular"0.8 ml
dexamethasone with both molarity 10-81.0 ml
serum human blood
or cattle100,0 ml
the rest,

while laundering produce medium containing all of the above components, except for the serum.

2. The method according to claim 1, characterized in that as the serum can be used fetal bovine serum or human serum, cord blood or human serum IV blood groups.



 

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