A skin cell dispersion and its use in the treatment of autologous burns

Abstract

The present invention relates to a skin cell dispersion liquid and its use in treating autogenous burn, and particularly, the present invention provides a skin cell dispersion liquid containing skin cells, growth factors and glutamine. The skin cell dispersion liquid of the present invention has excellent therapeutic effect on skin burn.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a skin cell dispersion and its use in the treatment of autologous burns. Background Technology

[0002] The skin serves as a barrier to maintain internal homeostasis and prevent the invasion of microorganisms and chemicals. Burns, especially large-area deep burns, can trigger numerous changes in various local and systemic systems, making them prone to infection, hindering wound healing, and easily leaving scars. If the exposed wound area is too large, bacteria can easily invade and multiply, causing electrolyte imbalances, anemia, high fever, sepsis, and other complications. Therefore, repairing large areas of skin loss is crucial.

[0003] Currently, commonly used methods for repairing wounds, especially large-area wounds, both domestically and internationally, mainly include microparticle skin grafting, large-sheet allograft skin grafting with small grafts, free skin grafting, Meek skin grafting, and autologous mesh skin grafting. However, these methods all have many drawbacks, such as allogeneic immune rejection, insufficient donor skin, slow skin healing speed and low healing rate, strong pain for patients, cumbersome operation, and long operation time. Patients with large-area burns cannot tolerate long-term surgery, thus limiting the effectiveness of existing technologies in burn treatment and causing suffering to patients.

[0004] Therefore, there is a need in this field to develop an effective drug for treating burns. Summary of the Invention

[0005] The purpose of this invention is to provide a skin cell dispersion that has excellent therapeutic effects on skin burns.

[0006] The first aspect of the present invention provides a skin cell dispersion containing skin cells, growth factors and glutamine.

[0007] Preferably, the skin cell dispersion is in the form of a cell suspension.

[0008] Preferably, the skin cell dispersion includes a skin cell dispersion for treating skin burns.

[0009] Preferably, the skin cell dispersion further includes a dispersion medium.

[0010] Preferably, the dispersion medium includes a cell dispersion medium.

[0011] Preferably, the cell dispersion medium comprises physiological saline or PBS buffered aqueous solution.

[0012] Preferably, the pH of the PBS buffer solution is 7.2-7.6, more preferably 7.4.

[0013] Preferably, the growth factor includes basic fibroblast growth factor.

[0014] Preferably, the growth factor includes human basic fibroblast growth factor.

[0015] Preferably, the growth factor includes recombinant human basic fibroblast growth factor.

[0016] Preferably, the glutamine includes L-glutamine.

[0017] Preferably, in the skin cell dispersion, the mass-volume concentration of the growth factor is 2-30 ng / ml, more preferably 5-20 ng / ml, even more preferably 5-15 ng / ml, even more preferably 8-12 ng / ml, and most preferably 10 ng / ml.

[0018] Preferably, in the skin cell dispersion, the mass-volume concentration of glutamine is 10-40 ng / ml, more preferably 15-35 ng / ml, even more preferably 20-30 ng / ml, even more preferably 23-27 ng / ml, and most preferably 25 ng / ml.

[0019] Preferably, the weight ratio of the growth factor to the glutamine is 1:0.5-8, more preferably 1:0.5-5, even more preferably 1:1-4, even more preferably 1:1.5-3.5, even more preferably 1:2-3, even more preferably 1:2.3-2.7, and even more preferably 1:2.5.

[0020] Preferably, the skin cells include skin cells obtained from skin dissociation.

[0021] Preferably, the dissociation is carried out in a dissociation solution.

[0022] In a preferred example, the area (cm²) of the skin 2 The volume ratio (ml) of the skin cell dispersion to the volume (cm) of the skin cell dispersion. 2 The ratio ( / ml) is 1:0.5-2, preferably 1:0.5-1.5, more preferably 1:0.8-1.2, and even more preferably 1:1.

[0023] Preferably, the skin cells are isolated or ex vivo skin cells.

[0024] Preferably, the skin comprises scaly skin.

[0025] Preferably, the skin cells include scabra cells.

[0026] Preferably, the skin cells comprise cells obtained from scalpel skin dissection.

[0027] Preferably, the skin includes normal skin.

[0028] Preferably, the skin cells include normal skin cells.

[0029] Preferably, the blade thickness skin includes normal blade thickness skin.

[0030] Preferably, the schizoderma cells comprise normal schizoderma cells.

[0031] Preferably, the skin cells include human skin cells or non-human mammalian skin cells.

[0032] Preferably, the skin includes human skin.

[0033] Preferably, the blade-thick skin includes human blade-thick skin.

[0034] Preferably, the skin cells include normal skin cells from the outer thigh.

[0035] Preferably, the skin cells comprise skin cells obtained by dissociation from normal lateral thigh skin.

[0036] Preferably, the skin cells comprise normal skin cells from a burn patient.

[0037] Preferably, the burn patients include patients with deep second-degree burns.

[0038] Preferably, the method for preparing skin cells includes:

[0039] (i) After taking the skin, the skin is dissociated in the dissociation solution to obtain the dissociated skin flap;

[0040] (ii) Separate the cells from the dissociated skin flap to obtain skin cells.

[0041] Preferably, in step (i), the skin is separated or excised skin.

[0042] Preferably, in step (i), the skin is obtained using a dermabrasion tool.

[0043] Preferably, in step (i), the skin comprises scaly skin.

[0044] Preferably, in step (i), the skin includes normal skin.

[0045] Preferably, the blade thickness skin includes normal blade thickness skin.

[0046] Preferably, in step (i), the skin includes human skin or non-human mammal skin.

[0047] Preferably, the non-human mammal includes pigs or primates.

[0048] Preferably, in step (i), the skin includes normal skin on the outer thigh.

[0049] Preferably, in step (i), the skin comprises the normal skin of a burn patient.

[0050] Preferably, the burn patients include patients with deep second-degree burns.

[0051] Preferably, in step (i), the skin is cleaned before the dissociation.

[0052] Preferably, in step (i), the skin is washed with physiological saline before dissociation.

[0053] Preferably, the dissociation solution includes a cell dissociation solution.

[0054] Preferably, in step (i), the dissociation solution includes trypsin and ethylenediaminetetraacetic acid.

[0055] Preferably, the trypsin is 0.2-1.5 wt% by weight, more preferably 0.2-1.0 wt%, more preferably 0.3-0.8 wt%, more preferably 0.4-0.6 wt%, and most preferably 0.5 wt%, based on the weight of the dissociation solution.

[0056] Preferably, the ethylenediaminetetraacetic acid is 0.02-0.15 wt% by weight, more preferably 0.02-0.1 wt%, more preferably 0.03-0.08 wt%, more preferably 0.04-0.06 wt%, and most preferably 0.05 wt% by weight of the dissociated liquid.

[0057] Preferably, in step (i), the dissociation temperature is 30-39°C, more preferably 35-37°C, and even more preferably 37°C.

[0058] Preferably, in step (i), the dissociation time is 10-30 min, more preferably 15-25 min, even more preferably 18-22 min, and most preferably 20 min.

[0059] Preferably, in step (i), the area (cm²) of the skin in step (i) 2 The volume ratio (ml) of the dissociation solution to the volume ratio (cm) of the dissociation solution 2 The ratio ( / ml) is 1:0.5-8, preferably 1:0.5-5, more preferably 1:1-4, more preferably 1:1.5-3.5, more preferably 1:2-3, more preferably 1:2.3-2.7, and more preferably 1:2.5.

[0060] Preferably, before step (ii), the disassembled leather pieces are cleaned with a cleaning solution.

[0061] Preferably, the cleaning solution comprises a PBS buffered aqueous solution containing a soybean trypsin inhibitor.

[0062] Preferably, the pH of the PBS buffer solution is 7.2-7.6, more preferably 7.4.

[0063] Preferably, the content of the soybean trypsin inhibitor is 0.02-0.5 wt%, more preferably 0.05-0.2 wt%, even more preferably 0.05-0.15 wt%, more preferably 0.08-0.12 wt%, and most preferably 0.1 wt%, based on the weight of the cleaning solution.

[0064] Preferably, the cleaning temperature is 30-39°C, more preferably 35-37°C, and most preferably 37°C.

[0065] Preferably, the cleaning solution is used to wash away any residual dissociation solution on the dissociated leather pieces.

[0066] Preferably, the area (cm²) of the skin in step (i) 2 The volume ratio (ml) of the cleaning solution to the volume ratio (cm) 2 The ratio ( / ml) is 1:0.5-8, preferably 1:0.5-5, more preferably 1:1-4, more preferably 1:1.5-3.5, more preferably 1:2-3, more preferably 1:2.3-2.7, and more preferably 1:2.5.

[0067] Preferably, in step (ii), the separation includes scraping.

[0068] Preferably, in step (ii), the separation is performed by scraping with the blade of a scalpel.

[0069] Preferably, the skin cells obtained in step (ii) are in the form of a cell suspension.

[0070] Preferably, the cell suspension includes a cell dispersion medium.

[0071] Preferably, step (ii) includes the step:

[0072] After separating the cells from the dissociated skin flap, the separated cells are mixed with a cell dispersion medium, filtered, and a skin cell suspension is obtained. The obtained skin cells exist in the form of a cell suspension.

[0073] Preferably, in the cell suspension, the average viable cell yield is 1.3 × 10⁻⁶. 6 pcs / cm2 -2.0×10 6 pcs / cm 2 Preferably 1.5×10 6 pcs / cm 2 -1.8×10 6 pcs / cm 2 .

[0074] Preferably, the epidermal cell content in the skin cell dispersion is 1.3 × 10⁻⁶. 6 pcs / cm 2 -2.0×10 6 pcs / cm 2 Preferably 1.5×10 6 pcs / cm 2 -1.8×10 6 pcs / cm 2 .

[0075] Preferably, the filter screen is an 80-120 mesh sieve, more preferably a 90-110 mesh sieve, and even more preferably a 100 mesh sieve.

[0076] Preferably, the cell dispersion medium comprises physiological saline or PBS buffered aqueous solution.

[0077] Preferably, the pH of the PBS buffer solution is 7.2-7.6, more preferably 7.4.

[0078] In a preferred example, the area (cm²) of the skin in step (i) 2 The volume ratio (ml) of the skin cell dispersion to the volume (cm) of the skin cell dispersion. 2 The ratio ( / ml) is 1:0.5-2, preferably 1:0.5-1.5, more preferably 1:0.8-1.2, and even more preferably 1:1.

[0079] In a preferred example, the area (cm²) of the skin in step (i) 2 The ratio of the mass (ng) of the growth factor to the mass (cm) of the growth factor 2 The ratio of / ng) is 1:2-35, preferably 1:3-30, even better 1:5-25, even better 1:5-20, even better 1:5-15, even better 1:8-12, even better 1:10.

[0080] In a preferred example, the area (cm²) of the skin in step (i) 2 The mass ratio (cm) of the glutamine to the mass (ng) of the glutamine 2 The ratio of / ng) is 1:5-45, preferably 1:10-40, even better 1:15-35, even better 1:20-30, even better 1:23-27, and even better 1:25.

[0081] In a second aspect, the present invention provides a method for preparing a skin cell dispersion as described in the first aspect of the present invention, the method comprising the steps of:

[0082] The skin cells were mixed with the growth factor and glutamine to obtain the skin cell dispersion.

[0083] Preferably, the method includes the following steps:

[0084] The skin cells, growth factors, glutamine, and dispersion medium are mixed to obtain the skin cell dispersion.

[0085] Preferably, the skin cells are prepared by the method described in the first aspect of the present invention.

[0086] Preferably, the method includes the following steps:

[0087] (i) After taking the skin, the skin is dissociated in the dissociation solution to obtain the dissociated skin flap;

[0088] (ii) Separate the cells from the dissociated skin flap to obtain skin cells;

[0089] (iii) The skin cells, growth factors and glutamine are mixed to obtain the cell dispersion.

[0090] Preferably, step (i) is as described in the first aspect of the invention.

[0091] Preferably, step (ii) is as described in the first aspect of the invention.

[0092] Preferably, the skin cells obtained in step (ii) are in the form of a cell suspension.

[0093] Preferably, the cell suspension includes a cell dispersion medium.

[0094] Preferably, step (ii) includes the step:

[0095] After separating the cells from the dissociated skin flap, the separated cells are mixed with a cell dispersion medium, filtered, and a skin cell suspension is obtained. The obtained skin cells exist in the form of a cell suspension.

[0096] Preferably, the filter screen is an 80-120 mesh sieve, more preferably a 90-110 mesh sieve, and even more preferably a 100 mesh sieve.

[0097] Preferably, the cell dispersion medium comprises physiological saline or PBS buffered aqueous solution.

[0098] Preferably, the pH of the PBS buffer solution is 7.2-7.6, more preferably 7.4.

[0099] Preferably, in step (iii), the skin cells, the growth factor, glutamine, and the cell dispersion medium are mixed to obtain the skin cell dispersion.

[0100] Preferably, step (iii) includes:

[0101] (iii) The skin cells, which are in the form of a cell suspension, are mixed with the growth factor and glutamine to obtain the skin cell dispersion.

[0102] Preferably, the method includes the following steps:

[0103] (i) Take a thick leather piece, place it in a dissociation solution to dissociate it, and obtain the dissociated leather piece;

[0104] (ii) After separating the cells from the dissociated skin flap, the separated cells are mixed with a cell dispersion medium and filtered to obtain a skin cell suspension, in which the skin cells exist in the form of a cell suspension;

[0105] (iii) The skin cell suspension is mixed with the growth factor and glutamine to obtain the skin cell dispersion.

[0106] Preferably, the method includes the following steps:

[0107] (i) Take a leather piece with a blade thickness of (1.8-2.2)cm×(1.8-2.2)cm, place the leather piece in a dissociation solution to dissociate it, and obtain the dissociated leather piece;

[0108] (ii) After separating the cells from the dissociated skin flap, the separated cells are mixed with 3.5-4.5 ml of cell dispersion medium, filtered, and a skin cell suspension is obtained. The obtained skin cells exist in the form of a cell suspension.

[0109] (iii) The skin cell suspension is mixed with the growth factor and glutamine to obtain the skin cell dispersion, wherein the mass-volume concentration of basic fibroblast growth factor in the skin cell dispersion is 8-12 ng / ml and the mass-volume concentration of glutamine is 23-27 ng / ml.

[0110] Preferably, the method includes the following steps:

[0111] (i) Take a (1.8-2.2)cm×(1.8-2.2)cm thick leather piece, place the leather piece in 9-11ml of dissociation solution and dissociate it at 35-37℃ for 18-22min to obtain the dissociated leather piece;

[0112] (ii) After separating the cells from the dissociated skin flap, the separated cells are mixed with 3.5-4.5 ml of cell dispersion medium and filtered through a 90-110 mesh sieve to obtain a skin cell suspension. The obtained skin cells exist in the form of a cell suspension.

[0113] (iii) The skin cell suspension is mixed with the growth factor and glutamine to obtain the skin cell dispersion, wherein the mass-volume concentration of basic fibroblast growth factor in the skin cell dispersion is 8-12 ng / ml and the mass-volume concentration of glutamine is 23-27 ng / ml.

[0114] Preferably, the method includes the following steps:

[0115] (i) Take a 2.0cm×2.0cm thick leather piece, place the leather piece in 10ml of dissociation solution and dissociate it at 37℃ for 20min to obtain the dissociated leather piece;

[0116] (ii) After separating the cells from the dissociated skin flap, the separated cells are mixed with 4 ml of cell dispersion medium and filtered through a 100-mesh sieve to obtain a skin cell suspension. The obtained skin cells exist in the form of a cell suspension.

[0117] (iii) The skin cell suspension is mixed with the growth factor and glutamine to obtain the skin cell dispersion, wherein the mass-volume concentration of basic fibroblast growth factor in the skin cell dispersion is 10 ng / ml and the mass-volume concentration of glutamine is 25 ng / ml.

[0118] In a third aspect, the present invention provides the use of the skin cell dispersion as described in the first aspect of the present invention for preparing a composition for treating skin burns.

[0119] Preferably, the burns include deep second-degree burns.

[0120] Preferably, the treatment includes autologous treatment.

[0121] Preferably, the composition is used to treat autologous skin burns.

[0122] Preferably, the treatment of skin burns includes promoting the healing of burned skin and / or reducing the pain of burned skin.

[0123] Preferably, the burned skin and the skin cells in the skin cell dispersion are derived from the same object (or patient).

[0124] Preferably, the skin cells in the skin cell dispersion are derived from normal skin cells of the same skin burn subject.

[0125] Preferably, the object includes a person.

[0126] Preferably, the dosage form of the composition is a liquid formulation, a solid formulation, or a semi-solid formulation.

[0127] Preferably, the dosage form of the composition is a topical skin preparation.

[0128] Preferably, the dosage form of the composition is a spray, a film-forming agent, an ointment, a cream, or a patch.

[0129] Preferably, the composition is a pharmaceutical composition.

[0130] Preferably, the composition further includes a pharmaceutically acceptable carrier.

[0131] In a fourth aspect, the present invention provides a composition comprising a skin cell dispersion as described in the first aspect of the present invention, and a pharmaceutically acceptable carrier.

[0132] Preferably, the dosage form of the composition is a liquid, solid, or semi-solid formulation.

[0133] Preferably, the dosage form of the composition is a topical skin preparation.

[0134] Preferably, the dosage form of the composition is a spray, a film-forming agent, an ointment, a cream, or a patch.

[0135] Preferably, the composition is a pharmaceutical composition.

[0136] Preferably, the composition further includes a pharmaceutically acceptable carrier.

[0137] In a fifth aspect, the present invention provides a method for treating skin burns by applying a skin cell dispersion as described in the first aspect of the present invention or a composition as described in the fourth aspect of the present invention to the burned subject, thereby treating the skin burns.

[0138] Preferably, the application is topical.

[0139] Preferably, the application is by spraying or smearing.

[0140] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0141] Figure 1 Cell morphology of cell suspension observed under an inverted phase-contrast microscope (×200).

[0142] Figure 2 To observe the cell morphology of the cell suspension after 24 hours of adherence and growth under an inverted phase-contrast microscope (×200).

[0143] Figure 3 Photographs showing the skin healing effect of the skin cell suspension prepared in Example 1 on burn skin 7 days after autologous treatment. Detailed Implementation

[0144] This invention, developed through extensive and in-depth research, unexpectedly produced a skin cell dispersion containing skin cells, growth factors, and glutamine. The skin cells in this dispersion can be directly derived from cells dissociated and collected from the skin (such as scalp lacerations), eliminating the need for in vitro cell culture. It can be directly sprayed onto autologous burn sites for treatment, significantly promoting rapid regeneration and healing of the burned skin, shortening healing time, and reducing pain. Based on this, the inventors completed this invention.

[0145] the term

[0146] As used herein, the term "comprising" includes not only closed definitions but also semi-closed and open definitions. In other words, the term includes "consisting of" and "substantially consisting of".

[0147] As used in this article, the terms "ethylenediaminetetraacetic acid" and "EDTA" are used interchangeably.

[0148] As used in this article, the term "recombinant human basic fibroblast growth factor" is abbreviated as rh-bFGF.

[0149] As used in this article, the term "basic fibroblast growth factor" is abbreviated as bFGF.

[0150] As used in this article, the English term “L-glutamine” is L-Glutamine, CAS number 56-85-9.

[0151] As used in this article, the English term "PBS" stands for phosphate buffered saline.

[0152] As used in this article, the terms "deep second-degree burn", "deep second-degree skin burn" and "deep second-degree burn" are used interchangeably.

[0153] As used in this article, the English term for "razor-thin graft" is razor-thin graft.

[0154] As used in this article, the English term "soybean trypsin inhibitor" is Trypsin inhibitor, soyabea.

[0155] Skin cell dispersion

[0156] This invention provides a skin cell dispersion that can be used to treat skin burns.

[0157] In a preferred embodiment of the invention, the skin cell dispersion contains skin cells, growth factors, and glutamine.

[0158] The skin cell dispersion described in this invention can be in the form of a cell suspension.

[0159] In a preferred embodiment of the present invention, the skin cell dispersion of the present invention further includes a dispersion medium.

[0160] Preferably, the dispersion medium includes a cell dispersion medium, for example, physiological saline or PBS buffered aqueous solution.

[0161] Preferably, the pH of the PBS buffer solution is 7.2-7.6, more preferably 7.4.

[0162] In a preferred embodiment of the present invention, the growth factor includes (but is not limited to) basic fibroblast growth factor. The basic fibroblast growth factor described in the present invention can be recombinant human basic fibroblast growth factor.

[0163] In a preferred embodiment of the invention, the glutamine comprises L-glutamine.

[0164] In a preferred embodiment of the present invention, the mass-volume concentration of the growth factor in the skin cell dispersion is 2-30 ng / ml, more preferably 5-20 ng / ml, more preferably 5-15 ng / ml, more preferably 8-12 ng / ml, and most preferably 10 ng / ml.

[0165] In a preferred embodiment of the present invention, the glutamine mass-volume concentration in the skin cell dispersion is 10-40 ng / ml, more preferably 15-35 ng / ml, more preferably 20-30 ng / ml, even more preferably 23-27 ng / ml, and most preferably 25 ng / ml.

[0166] Preferably, the weight ratio of the growth factor to the glutamine is 1:0.5-8, more preferably 1:0.5-5, even more preferably 1:1-4, even more preferably 1:1.5-3.5, even more preferably 1:2-3, even more preferably 1:2.3-2.7, and even more preferably 1:2.5.

[0167] In a preferred embodiment of the invention, the skin cells comprise skin cells obtained from skin dissociation.

[0168] In a preferred example, the area (cm²) of the skin 2 The volume ratio (ml) of the skin cell dispersion to the volume (cm) of the skin cell dispersion. 2 The ratio ( / ml) is 1:0.2-6, preferably 1:0.5-2, more preferably 1:0.5-1.5, even better 1:0.8-1.2, and optimally 1:1.

[0169] In a preferred embodiment of the present invention, the skin cells are isolated or ex vivo skin cells.

[0170] In a preferred embodiment of the invention, the skin comprises scabra. The skin cells may include scabra cells.

[0171] In a preferred embodiment of the invention, the skin comprises normal skin. The skin cells comprise normal skin cells.

[0172] Preferably, the blade thickness skin includes normal blade thickness skin.

[0173] In a preferred embodiment of the invention, the skin cells include human skin cells or non-human mammalian (such as pig) skin cells.

[0174] Preferably, the skin includes human skin.

[0175] Preferably, the blade-thick skin includes human blade-thick skin.

[0176] Preferably, the skin cells include normal skin cells from the outer thigh.

[0177] Preferably, the skin cells comprise normal skin cells from a burn patient.

[0178] Preferably, the burn patients include patients with deep second-degree burns.

[0179] In a preferred embodiment of the present invention, the method for preparing skin cells includes:

[0180] (i) After taking the skin, the skin is dissociated in the dissociation solution to obtain the dissociated skin flap;

[0181] (ii) Separate the cells from the dissociated skin flap to obtain skin cells.

[0182] In a preferred embodiment of the invention, in step (i), the skin is separated or excised skin. Preferably, the skin includes normal skin.

[0183] In a preferred embodiment of the invention, in step (i), the skin comprises scabbard. Preferably, the scabbard comprises normal scabbard.

[0184] In a preferred embodiment of the invention, in step (i), the skin comprises human skin or non-human mammal skin. Preferably, the non-human mammal comprises pigs or primates.

[0185] In a preferred embodiment of the invention, in step (i), the skin comprises normal skin on the outer thigh.

[0186] In a preferred embodiment of the invention, in step (i), the skin comprises the normal skin of a burn patient. Preferably, the burn patient comprises a patient with deep second-degree burns.

[0187] In a preferred embodiment of the invention, in step (i), the skin is cleaned before dissociation. For example, the skin can be cleaned with physiological saline before dissociation.

[0188] In a preferred embodiment of the present invention, in step (i), the dissociation solution includes a cell dissociation solution.

[0189] Preferably, in step (i), the dissociation solution includes trypsin and ethylenediaminetetraacetic acid.

[0190] Preferably, the trypsin is 0.2-1.5 wt% by weight, more preferably 0.2-1.0 wt%, more preferably 0.3-0.8 wt%, more preferably 0.4-0.6 wt%, and most preferably 0.5 wt%, based on the weight of the dissociation solution.

[0191] Preferably, the ethylenediaminetetraacetic acid is 0.02-0.15 wt% by weight, more preferably 0.02-0.1 wt%, more preferably 0.03-0.08 wt%, more preferably 0.04-0.06 wt%, and most preferably 0.05 wt% by weight of the dissociated liquid.

[0192] Preferably, in step (i), the dissociation temperature is 30-39°C, more preferably 35-37°C, and even more preferably 37°C.

[0193] Preferably, in step (i), the dissociation time is 10-30 min, more preferably 15-25 min, even more preferably 18-22 min, and most preferably 20 min.

[0194] In a preferred embodiment of the present invention, in step (i), the area (cm²) of the skin... 2 The volume ratio (ml) of the dissociation solution to the volume ratio (cm) of the dissociation solution 2 The ratio ( / ml) is 1:0.5-8, preferably 1:0.5-5, more preferably 1:1-4, more preferably 1:1.5-3.5, more preferably 1:2-3, more preferably 1:2.3-2.7, and more preferably 1:2.5.

[0195] In a preferred embodiment of the invention, the disassembled leather pieces are cleaned with a cleaning solution before step (ii).

[0196] Preferably, the cleaning solution comprises a PBS buffered aqueous solution containing a soybean trypsin inhibitor.

[0197] Preferably, the pH of the PBS buffer solution is 7.2-7.6, more preferably 7.4.

[0198] Preferably, the content of the soybean trypsin inhibitor is 0.02-0.5 wt%, more preferably 0.05-0.2 wt%, even more preferably 0.05-0.15 wt%, more preferably 0.08-0.12 wt%, and most preferably 0.1 wt%, based on the weight of the cleaning solution.

[0199] Preferably, the cleaning temperature is 30-39°C, more preferably 35-37°C, and most preferably 37°C.

[0200] Preferably, the cleaning solution is used to wash away any residual dissociation solution on the dissociated leather pieces.

[0201] Preferably, the area (cm²) of the skin in step (i) 2 The volume ratio (ml) of the cleaning solution to the volume ratio (cm) 2 The ratio ( / ml) is 1:0.5-8, preferably 1:0.5-5, more preferably 1:1-4, more preferably 1:1.5-3.5, more preferably 1:2-3, more preferably 1:2.3-2.7, and more preferably 1:2.5.

[0202] In a preferred embodiment of the invention, in step (ii), the separation includes scraping. The separation can be performed by scraping with the blade of a scalpel.

[0203] In a preferred embodiment of the invention, the skin cells obtained in step (ii) are present in the form of a cell suspension.

[0204] Preferably, the cell suspension includes a cell dispersion medium.

[0205] Preferably, step (ii) includes the step:

[0206] After separating the cells from the dissociated skin flap, the separated cells are mixed with a cell dispersion medium, filtered, and a skin cell suspension is obtained. The obtained skin cells exist in the form of a cell suspension.

[0207] Preferably, the filter screen is an 80-120 mesh sieve, more preferably a 90-110 mesh sieve, and even more preferably a 100 mesh sieve.

[0208] Preferably, the cell dispersion medium comprises physiological saline or PBS buffered aqueous solution.

[0209] Preferably, the pH of the PBS buffer solution is 7.2-7.6, more preferably 7.4.

[0210] In a preferred embodiment of the invention, the skin area (cm²) in step (i) 2 The volume ratio (ml) of the skin cell dispersion to the volume (cm) of the skin cell dispersion. 2 The ratio ( / ml) is 1:0.5-2, preferably 1:0.5-1.5, more preferably 1:0.8-1.2, and even more preferably 1:1.

[0211] In a preferred example, the area (cm²) of the skin in step (i) 2 The ratio of the mass (ng) of the growth factor to the mass (cm) of the growth factor 2 The ratio of / ng) is 1:2-35, preferably 1:3-30, even better 1:5-25, even better 1:5-20, even better 1:5-15, even better 1:8-12, even better 1:10.

[0212] In a preferred example, the area (cm²) of the skin in step (i) 2 The mass ratio (cm) of the glutamine to the mass (ng) of the glutamine 2 The ratio of / ng) is 1:5-45, preferably 1:10-40, even better 1:15-35, even better 1:20-30, even better 1:23-27, and even better 1:25.

[0213] method

[0214] This invention provides a method for preparing the skin cell dispersion described herein, the method comprising the steps of:

[0215] The skin cells were mixed with the growth factor and glutamine to obtain the skin cell dispersion.

[0216] In a preferred embodiment of the present invention, the method includes the steps of:

[0217] The skin cells, growth factors, glutamine, and dispersion medium are mixed to obtain the skin cell dispersion.

[0218] In a preferred embodiment of the invention, the skin cells are prepared by the method described in the first aspect of the invention.

[0219] In a preferred embodiment of the present invention, the method includes the steps of:

[0220] (i) After taking the skin, the skin is dissociated in the dissociation solution to obtain the dissociated skin flap;

[0221] (ii) Separate the cells from the dissociated skin flap to obtain skin cells;

[0222] (iii) The skin cells, growth factors and glutamine are mixed to obtain the cell dispersion.

[0223] In a preferred embodiment of the invention, step (i) is as described in the first aspect of the invention.

[0224] In a preferred embodiment of the invention, step (ii) is as described in the first aspect of the invention.

[0225] In a preferred embodiment of the invention, the skin cells obtained in step (ii) are present in the form of a cell suspension.

[0226] Preferably, the cell suspension includes a cell dispersion medium.

[0227] Preferably, step (ii) includes the step:

[0228] After separating the cells from the dissociated skin flap, the separated cells are mixed with a cell dispersion medium, filtered, and a skin cell suspension is obtained. The obtained skin cells exist in the form of a cell suspension.

[0229] Preferably, the filter screen is an 80-120 mesh sieve, more preferably a 90-110 mesh sieve, and even more preferably a 100 mesh sieve.

[0230] Preferably, the cell dispersion medium comprises physiological saline or PBS buffered aqueous solution.

[0231] Preferably, the pH of the PBS buffer solution is 7.2-7.6, more preferably 7.4.

[0232] Preferably, in step (iii), the skin cells, the growth factor, glutamine, and the cell dispersion medium are mixed to obtain the skin cell dispersion.

[0233] In a preferred embodiment of the present invention, step (iii) includes:

[0234] (iii) The skin cells, which are in the form of a cell suspension, are mixed with the growth factor and glutamine to obtain the skin cell dispersion.

[0235] Typically, the method includes the steps of:

[0236] (i) Take a thick leather piece, place it in a dissociation solution to dissociate it, and obtain the dissociated leather piece;

[0237] (ii) After separating the cells from the dissociated skin flap, the separated cells are mixed with a cell dispersion medium and filtered to obtain a skin cell suspension, in which the skin cells exist in the form of a cell suspension;

[0238] (iii) The skin cell suspension is mixed with the growth factor and glutamine to obtain the skin cell dispersion.

[0239] Preferably, the method includes the following steps:

[0240] (i) Take a leather piece with a blade thickness of (1.8-2.2)cm×(1.8-2.2)cm, place the leather piece in a dissociation solution to dissociate it, and obtain the dissociated leather piece;

[0241] (ii) After separating the cells from the dissociated skin flap, the separated cells are mixed with 3.5-4.5 ml of cell dispersion medium, filtered, and a skin cell suspension is obtained. The obtained skin cells exist in the form of a cell suspension.

[0242] (iii) The skin cell suspension is mixed with the growth factor and glutamine to obtain the skin cell dispersion, wherein the mass-volume concentration of basic fibroblast growth factor in the skin cell dispersion is 8-12 ng / ml and the mass-volume concentration of glutamine is 23-27 ng / ml.

[0243] Typically, the method includes the steps of:

[0244] (i) Take a (1.8-2.2)cm×(1.8-2.2)cm thick leather piece, place the leather piece in 9-11ml of dissociation solution and dissociate it at 35-37℃ for 18-22min to obtain the dissociated leather piece;

[0245] (ii) After separating the cells from the dissociated skin flap, the separated cells are mixed with 3.5-4.5 ml of cell dispersion medium and filtered through a 90-110 mesh sieve to obtain a skin cell suspension. The obtained skin cells exist in the form of a cell suspension.

[0246] (iii) The skin cell suspension is mixed with the growth factor and glutamine to obtain the skin cell dispersion, wherein the mass-volume concentration of basic fibroblast growth factor in the skin cell dispersion is 8-12 ng / ml and the mass-volume concentration of glutamine is 23-27 ng / ml.

[0247] use

[0248] The present invention also provides the use of the skin cell dispersion as described herein for preparing a composition for treating skin burns.

[0249] In a preferred embodiment of the invention, the burns include deep second-degree burns.

[0250] In a preferred embodiment of the invention, the treatment includes autologous treatment.

[0251] In this invention, it should be understood that autologous treatment refers to the use of skin cell dispersions and burnt skin as the same object (or patient), that is, skin cell dispersions prepared by separating skin cells from the skin of the object (or patient) with burnt skin are used to treat the same object (or patient) with burnt skin.

[0252] In a preferred embodiment of the invention, the composition is used to treat autologous skin burns.

[0253] In a preferred embodiment of the invention, the treatment of skin burns includes (but is not limited to) promoting the healing of burned skin and / or reducing the pain of burned skin.

[0254] In a preferred embodiment of the invention, the burned skin and the skin cells in the skin cell dispersion are derived from the same object (or patient).

[0255] Preferably, the skin cells in the skin cell dispersion are derived from normal skin cells of the same skin burn subject.

[0256] Preferably, the object includes a person.

[0257] In a preferred embodiment of the present invention, the dosage form of the composition is a liquid formulation, a solid formulation, or a semi-solid formulation.

[0258] In a preferred embodiment of the present invention, the dosage form of the composition is a topical skin preparation.

[0259] In a preferred embodiment of the present invention, the dosage form of the composition is a spray, a film-forming agent, an ointment, a cream, or a patch.

[0260] Preferably, the composition is a pharmaceutical composition.

[0261] Preferably, the composition further includes a pharmaceutically acceptable carrier.

[0262] Composition

[0263] The compositions described in this invention are preferably pharmaceutical compositions. The compositions described in this invention may also include a pharmaceutically acceptable carrier.

[0264] As used herein, "pharmaceutically acceptable carrier" refers to one or more compatible solid, semi-solid, liquid, or gel fillers that are suitable for human or animal use and must have sufficient purity and sufficiently low toxicity. "Compatibility" refers to the ability of the components in a pharmaceutical composition and the active ingredient of the drug, as well as their interactions, to not significantly reduce the efficacy of the drug.

[0265] It should be understood that, in this invention, there are no particular limitations on the pharmaceutically acceptable carriers used. Materials commonly used in the art can be selected, or they can be prepared using conventional methods or purchased from the market. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium dodecyl sulfate), buffers, chelating agents, thickeners, pH adjusters, transdermal penetration enhancers, colorants, flavoring agents, stabilizers, antioxidants, preservatives, antibacterial agents, pyrogen-free water, etc.

[0266] In this invention, the dosage form of the composition includes, but is not limited to, a topical skin preparation. The topical skin preparations described in this invention are preferably preparations for use on burned skin.

[0267] Typically, the dosage form of the composition includes, but is not limited to, liquid, solid, or semi-solid formulations. For example, the dosage form of the composition may be a spray, film-forming agent, cream, lotion, or patch.

[0268] Pharmaceutical formulations should be matched to the route of administration, preferably for topical application, whereby a therapeutically effective amount of the drug is administered to the desired subject (e.g., human or non-human mammal). As used herein, the term "therapeutically effective amount" refers to an amount that is functionally or activityily produced in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "therapeutically effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and whether it is used in combination with other drugs.

[0269] The safe and effective daily dosage of the active ingredient should take into account factors such as the route of administration and the health status of the patient / animal, all of which are within the skill range of a skilled physician / researcher.

[0270] The superior technical effects of this invention include:

[0271] 1. This invention develops a skin cell dispersion with excellent therapeutic effect on skin burns. The skin cells in the dispersion can be directly derived from cells dissociated and collected from the skin (such as scald skin), eliminating the need for in vitro cell culture. It can be directly sprayed onto the burn site for treatment, improving treatment efficiency, reducing operation time, reducing surgical pain, and lowering costs. It is simple and convenient to operate, and can significantly promote rapid regeneration and healing of the burned skin, shorten the skin healing time, and reduce pain.

[0272] 2. The skin cell dispersion of the present invention can significantly increase the amount of cell dispersion prepared by skin harvesting per unit area applied to the burned skin area, enabling the treatment of large-area burns and wounds with a small donor area. It can be applied to patients with large-area burns, thereby reducing the amount of skin harvested by the patient, reducing the patient's pain, and significantly improving the treatment efficiency.

[0273] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0274] Example 1

[0275] The preparation of skin cell suspension is as follows:

[0276] 1. Preparation of dissociation solution and washing buffer

[0277] 1.1 Preparation of the dissociation solution:

[0278] Trypsin and ethylenediaminetetraacetic acid (EDTA) were mixed in physiological saline to obtain a dissociation solution, wherein the weight percentage of trypsin was 0.5 wt% and the weight percentage of EDTA was 0.05 wt%, based on the weight of the dissociation solution.

[0279] 1.2 Preparation of washing buffer:

[0280] Soybean trypsin inhibitor was mixed in PBS 7.4 buffer solution to obtain a washing buffer, wherein the weight percentage of soybean trypsin inhibitor was 0.1 wt%, based on the weight of the washing buffer.

[0281] 2. Preparation of skin cell suspension, as follows:

[0282] (1) Using a skin grafting knife, a 2cm×2cm non-wound skin graft was taken from the normal outer thigh of a patient with deep second-degree burns. After being thoroughly cleaned with physiological saline, the skin graft was placed in 10ml of dissociation solution and dissociated at 37℃ for 20min to obtain the dissociated skin graft.

[0283] (2) Use sterile tweezers to remove the dissociated skin piece and immerse it in 10 ml of 37°C washing buffer to wash away the residual dissociation solution and obtain the washed skin piece.

[0284] (3) Place the cleaned skin graft in a sterile tray, fix the skin graft with medical forceps, scrape off the cells with the blade of a scalpel, then rinse the scalpel and tray with 4 ml of PBS 7.4 buffer solution, collect the obtained cell suspension in one corner of the tray, and use a syringe and blunt needle to draw the cell suspension into the syringe.

[0285] (4) After filtering the cell suspension in the syringe obtained in step (3) through a 100-mesh sieve, the filtered cell suspension is obtained.

[0286] (5) The filtered cell suspension obtained in step (4) is mixed with recombinant human basic fibroblast growth factor (rh-bFGF) and L-glutamine to obtain a skin cell suspension, wherein the mass-volume concentration of recombinant human basic fibroblast growth factor (rh-bFGF) in the skin cell suspension is 10 ng / ml and the mass-volume concentration of L-glutamine is 25 ng / ml.

[0287] Effect Measurement

[0288] The cell morphology in the filtered cell suspension prepared in step (4) is as follows: Figure 1 As shown, from Figure 1 As can be seen, the cells in the filtered cell suspension were in good condition, and the average viable cell yield was measured to be 1.62 × 10⁻⁶. 6 / cm 2 The vitality rate is 85%.

[0289] The cell morphology of the filtered cell suspension prepared in step (4) after 24 hours of adherence and growth in serum-free culture medium is as follows. Figure 2 As shown, from Figure 2 As can be seen, the cells adhere to the wall and aggregate in a clonal manner, exhibiting excellent adhesion growth characteristics, thus demonstrating excellent regenerative and repair capabilities at the site of burns.

[0290] Example 2

[0291] Example 2 is the same as Example 1, except that: "2. Preparation of skin cell suspension, as follows":

[0292] (1) Using a skin grafting knife, a 2cm×2cm full-thickness skin graft was taken from the normal outer thigh of a patient with deep second-degree burns. After being thoroughly cleaned with physiological saline, the skin graft was placed in 10ml of dissociation solution and dissociated at 37℃ for 20min to obtain the dissociated skin graft.

[0293] (2) Use sterile tweezers to remove the dissociated skin piece and immerse it in 10 ml of 37°C washing buffer to wash away the residual dissociation solution and obtain the washed skin piece.

[0294] (3) Place the cleaned skin graft in a sterile tray, fix the skin graft with medical forceps, scrape off the cells with the blade of a scalpel, then rinse the scalpel and tray with 4 ml of PBS 7.4 buffer solution, collect the obtained cell suspension in one corner of the tray, and use a syringe and blunt needle to draw the cell suspension into the syringe.

[0295] (4) After filtering the cell suspension in the syringe obtained in step (3) through a 100-mesh sieve, the filtered cell suspension is obtained.

[0296] (5) The filtered cell suspension obtained in step (4) is mixed with recombinant human basic fibroblast growth factor (rh-bFGF) and L-glutamine to obtain a skin cell suspension, wherein the mass-volume concentration of recombinant human basic fibroblast growth factor (rh-bFGF) in the skin cell suspension is 10 ng / ml and the mass-volume concentration of L-glutamine is 25 ng / ml.

[0297] Example 3

[0298] Example 3 is the same as Example 1, except that: "2. Preparation of skin cell suspension, as follows":

[0299] (1) Using a skin grafting knife, a 2cm×2cm non-wound skin graft was taken from the normal outer thigh of a patient with deep second-degree burns. After being thoroughly cleaned with physiological saline, the skin graft was placed in 10ml of dissociation solution and dissociated at 37℃ for 20min to obtain the dissociated skin graft.

[0300] (2) Use sterile tweezers to remove the dissociated skin piece and immerse it in 10 ml of 37°C washing buffer to wash away the residual dissociation solution and obtain the washed skin piece.

[0301] (3) Place the cleaned skin graft in a sterile tray, fix the skin graft with medical forceps, scrape off the cells with the blade of a scalpel, then rinse the scalpel and tray with 4 ml of PBS 7.4 buffer solution, collect the obtained cell suspension in one corner of the tray, and use a syringe and blunt needle to draw the cell suspension into the syringe.

[0302] (4) After filtering the cell suspension in the syringe obtained in step (3) through a 100-mesh sieve, the filtered cell suspension is obtained.

[0303] (5) The filtered cell suspension obtained in step (4) is mixed with L-glutamine to obtain a skin cell suspension, wherein the mass-volume concentration of L-glutamine in the skin cell suspension is 50 ng / ml.

[0304] Example 4

[0305] Example 4 is the same as Example 1, except that: "2. Preparation of skin cell suspension, as follows":

[0306] (1) Using a skin grafting knife, a 2cm×2cm non-wound skin graft was taken from the normal outer thigh of a patient with deep second-degree burns. After being thoroughly cleaned with physiological saline, the skin graft was placed in 10ml of dissociation solution and dissociated at 37℃ for 20min to obtain the dissociated skin graft.

[0307] (2) Use sterile tweezers to remove the dissociated skin piece and immerse it in 10 ml of 37°C washing buffer to wash away the residual dissociation solution and obtain the washed skin piece.

[0308] (3) Place the cleaned skin graft in a sterile tray, fix the skin graft with medical forceps, scrape off the cells with the blade of a scalpel, then rinse the scalpel and tray with 4 ml of PBS 7.4 buffer solution, collect the obtained cell suspension in one corner of the tray, and use a syringe and blunt needle to draw the cell suspension into the syringe.

[0309] (4) After filtering the cell suspension in the syringe obtained in step (3) through a 100-mesh sieve, the filtered cell suspension is obtained.

[0310] (5) The filtered cell suspension obtained in step (4) is mixed with recombinant human basic fibroblast growth factor (rh-bFGF) to obtain a skin cell suspension, wherein the mass-volume concentration of recombinant human basic fibroblast growth factor (rh-bFGF) in the skin cell suspension is 20 ng / ml.

[0311] Example 5

[0312] Example 5 is the same as Example 1, except that: "2. Preparation of skin cell suspension, as follows":

[0313] (1) Using a skin grafting knife, a 2cm×2cm non-wound skin graft was taken from the normal outer thigh of a patient with deep second-degree burns. After being thoroughly cleaned with physiological saline, the skin graft was placed in 10ml of dissociation solution and dissociated at 37℃ for 20min to obtain the dissociated skin graft.

[0314] (2) Use sterile tweezers to remove the dissociated skin piece and immerse it in 10 ml of 37°C washing buffer to wash away the residual dissociation solution and obtain the washed skin piece.

[0315] (3) Place the cleaned skin graft in a sterile tray, fix the skin graft with medical forceps, scrape off the cells with the blade of a scalpel, then rinse the scalpel and tray with 4 ml of PBS 7.4 buffer solution, collect the obtained cell suspension in one corner of the tray, and use a syringe and blunt needle to draw the cell suspension into the syringe.

[0316] (4) After filtering the cell suspension in the syringe obtained in step (3) through a 100-mesh sieve, the filtered cell suspension is obtained.

[0317] (5) The filtered cell suspension obtained in step (4) is mixed with recombinant human basic fibroblast growth factor (rh-bFGF) and L-glutamine to obtain a skin cell suspension, wherein the mass-volume concentration of recombinant human basic fibroblast growth factor (rh-bFGF) in the skin cell suspension is 10 ng / ml and the mass-volume concentration of L-glutamine is 5 ng / ml.

[0318] Example 6

[0319] Example 6 is the same as Example 1, except that: "2. Preparation of skin cell suspension, as follows":

[0320] (1) Using a skin grafting knife, a 2cm×2cm non-wound skin graft was taken from the normal outer thigh of a patient with deep second-degree burns. After being thoroughly cleaned with physiological saline, the skin graft was placed in 10ml of dissociation solution and dissociated at 37℃ for 20min to obtain the dissociated skin graft.

[0321] (2) Use sterile tweezers to remove the dissociated skin piece and immerse it in 10 ml of 37°C washing buffer to wash away the residual dissociation solution and obtain the washed skin piece.

[0322] (3) Place the cleaned skin graft in a sterile tray, fix the skin graft with medical forceps, scrape off the cells with the blade of a scalpel, then rinse the scalpel and tray with 4 ml of PBS 7.4 buffer solution, collect the obtained cell suspension in one corner of the tray, and use a syringe and blunt needle to draw the cell suspension into the syringe.

[0323] (4) After filtering the cell suspension in the syringe obtained in step (3) through a 100-mesh sieve, the filtered cell suspension is obtained.

[0324] (5) The filtered cell suspension obtained in step (4) is mixed with recombinant human epidermal growth factor (rh-EGF) and L-glutamine to obtain a skin cell suspension, wherein the mass-volume concentration of recombinant human epidermal growth factor (rh-EGF) in the skin cell suspension is 10 ng / ml and the mass-volume concentration of L-glutamine is 25 ng / ml.

[0325] Example 7

[0326] Example 7 is the same as Example 1, except that: "2. Preparation of skin cell suspension, as follows":

[0327] (1) Using a skin grafting knife, a 2cm×2cm non-wound skin graft was taken from the normal outer thigh of a patient with deep second-degree burns. After being thoroughly cleaned with physiological saline, the skin graft was placed in 10ml of dissociation solution and dissociated at 37℃ for 20min to obtain the dissociated skin graft.

[0328] (2) Use sterile tweezers to remove the dissociated skin piece and immerse it in 10 ml of 37°C washing buffer to wash away the residual dissociation solution and obtain the washed skin piece.

[0329] (3) Place the cleaned skin graft in a sterile tray, fix the skin graft with medical forceps, scrape off the cells with the blade of a scalpel, then rinse the scalpel and tray with 4 ml of PBS 7.4 buffer solution, collect the obtained cell suspension in one corner of the tray, and use a syringe and blunt needle to draw the cell suspension into the syringe.

[0330] (4) After filtering the cell suspension in the syringe obtained in step (3) through a 100-mesh sieve, the filtered cell suspension is obtained, which is the skin cell suspension.

[0331] Example of effect

[0332] The skin cell suspensions prepared in Examples 1-7 were examined for use in autologous treatment of burned skin in patients with deep second-degree burns.

[0333] 1. Treatment methods

[0334] The methods for using the skin cell suspensions prepared in Examples 1-7 for autologous treatment of burned skin in patients with deep second-degree burns are as follows:

[0335] After debridement of the burn wound, skin cell suspensions prepared from normal, non-wounded skin grafts from the lateral thigh of the same patient (Examples 1-7) were sprayed onto the autologous burn skin of the patient with deep second-degree burns. The spraying volume was based on the skin cell suspension prepared from a 1cm × 1cm skin graft area, which was evenly sprayed over an 80cm area. 2 Spray the autologous burn skin surface and then cover and bandage it with sterile dressing.

[0336] 2. Evaluation Indicators

[0337] 1. Evaluation of wound healing rate

[0338] Wound healing evaluation criteria: The wound surface is epithelialized, there is no exudate, and no drainage or dressing is required.

[0339] The wound healing rate of the skin cell suspensions prepared in Examples 1-7 on day 7 after autologous treatment of burn skin in patients with deep second-degree burns was evaluated.

[0340] Wound healing rate = (initial wound contour area - postoperative unhealed wound contour area) / initial wound contour area × 100%.

[0341] Before and 7 days after autologous treatment of deep second-degree burn patients with skin cell suspensions in each embodiment, the wound surface was covered with sterile plastic film and its outline was drawn. Then, the drawn sterile plastic film was scanned into a computer, and the wound length (cm), width (cm), and outline area (cm²) were calculated and analyzed using image analysis software (Adobe Photoshop 7.0 and ImageJ version 1.46j). 2 The study aimed to evaluate the healing effect of skin cell suspensions from each embodiment on burn wounds in patients with autologous deep second-degree burns.

[0342] Table 1 shows the wound healing rates of the skin cell suspensions prepared in Examples 1-7 after 7 days of autologous treatment of burn skin in patients with deep second-degree burns:

[0343] Table 1. Wound healing rate of skin cell suspensions prepared in Examples 1-7 after autologous treatment of burn skin in patients with deep second-degree burns (n=10)

[0344]

[0345] As shown in Table 1, the skin cell suspension prepared in this embodiment exhibits excellent regenerative and healing capabilities for burned skin. Basic fibroblast growth factor and glutamine synergistically enhance the therapeutic effect of split-thickness dermatome on burned skin. In particular, the skin cell suspension prepared in Example 1 significantly promotes the regeneration and healing of burned skin, thus achieving a superior therapeutic effect. The effect photos of the skin cell suspension prepared in Example 1 on autologous burned skin are shown below. Figure 3 As shown. From Figure 3 As can be seen, the skin cell suspension prepared in Example 1 significantly healed the burned skin after autologous treatment. Therefore, the skin cell suspension prepared in Example 1 can significantly promote the healing of burned skin, thus having a better therapeutic effect on burned skin.

[0346] 2. Pain assessment

[0347] Pain in patients with deep second-degree burns is assessed using the National Scorecard (NRS) score. The NRS scoring criteria are as follows:

[0348] The Numerical Rating Scale (NRS) uses a 10cm long pain scale, where 0 represents no pain and 10 represents the most painful. Patients choose one number from these 11 numbers to represent their pain level.

[0349] NRS pain rating scale (0-10 points):

[0350]

[0351] 0 points: Painless;

[0352] 1-3 points: Mild pain, sleep unaffected;

[0353] 4-6 points: Moderate pain, sleep disturbance;

[0354] 7-9 points: Severe pain, seriously affecting sleep;

[0355] 10 / 10: Extreme pain.

[0356] The higher the score, the more severe the pain.

[0357] Table 2 shows the pain subjective rating (NRS score) of patients with deep second-degree burns 7 days after autologous treatment of the skin cell suspensions prepared in Examples 1-7 with the burned skin.

[0358] Table 2 shows the subjective pain scores of patients (n=10) on the 7th day after autologous treatment of the burned skin of patients with deep second-degree burns using the skin cell suspensions prepared in Examples 1-7.

[0359] Skin cell suspension prepared in Example 1 2 Skin cell suspension prepared in Example 2 7 Skin cell suspension prepared in Example 3 5 Skin cell suspension prepared in Example 4 4 Skin cell suspension prepared in Example 5 5 Skin cell suspension prepared in Example 6 4 Skin cell suspension prepared in Example 7 6

[0360] As can be seen from Table 2, the skin cell suspension prepared in this embodiment can treat the pain caused by burned skin, thus having an excellent therapeutic effect on burns.

[0361] The above description is an implementation scheme designed for one case of the present invention. It should be noted that for those skilled in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. A skin cell dispersion, characterized in that, The skin cell dispersion contains skin cells, basic fibroblast growth factor, and glutamine; The skin cell dispersion is prepared by the following method: (i) Take a thick leather piece and place it in a dissociation solution to dissociate it, thus obtaining the dissociated leather piece; (ii) After separating the cells from the dissociated skin flap, the separated cells are mixed with a cell dispersion medium, filtered, and a skin cell suspension is obtained. The obtained skin cells exist in the form of a cell suspension. (iii) The skin cell suspension is mixed with the basic fibroblast growth factor and glutamine to obtain the skin cell dispersion, wherein the basic fibroblast growth factor has a mass-volume concentration of 8-12 ng / ml and the glutamine has a mass-volume concentration of 23-27 ng / ml in the skin cell dispersion. The cell dispersion medium is physiological saline or PBS buffered aqueous solution; The glutamine is L-glutamine.

2. The skin cell dispersion as described in claim 1, characterized in that, In the skin cell dispersion, the basic fibroblast growth factor has a mass-to-volume concentration of 10 ng / ml; and In the skin cell dispersion, the mass-volume concentration of glutamine is 25 ng / ml.

3. The skin cell dispersion as described in claim 1, characterized in that, In step (i), the dissociation solution includes trypsin and ethylenediaminetetraacetic acid.

4. The skin cell dispersion as described in claim 3, characterized in that, The trypsin comprises 0.3-0.8 wt% of the weight of the dissociation solution; and / or The weight percentage of the ethylenediaminetetraacetic acid is 0.03-0.08 wt%, based on the weight of the dissociation solution.

5. The skin cell dispersion as described in claim 1, characterized in that, In step (i), the ratio of the area of ​​the blade thickness to the volume of the dissociation liquid is 1 cm. 2 : (2-3) ml.

6. The use of the skin cell dispersion as described in claim 1, characterized in that, Used to prepare a composition for treating skin burns.

7. The use as described in claim 6, characterized in that, The treatment of skin burns includes promoting the healing of burned skin and / or reducing the pain of burned skin.

8. The use as described in claim 6, characterized in that, The burns include deep second-degree burns; The composition is a topical skin preparation.

9. The use as described in claim 6, characterized in that, The dosage form of the composition is a spray, a film, an ointment, a cream, or a patch.

10. A composition, characterized in that, The composition comprises the skin cell dispersion as described in claim 1; and a pharmaceutically acceptable carrier.

11. The composition according to claim 10, characterized in that, The composition is a pharmaceutical composition.

12. The composition according to claim 10, characterized in that, The composition also includes a pharmaceutically acceptable carrier.

13. The composition of claim 10, characterized in that, The composition is a topical skin preparation.

14. The composition of claim 10, characterized in that, The dosage form of the composition is a spray, a film, an ointment, a cream, or a patch.

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