CN103037871A - Method for preparing autologous skin cell agent for alleviating or treating skin defects - Google Patents
Method for preparing autologous skin cell agent for alleviating or treating skin defects Download PDFInfo
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Abstract
The present invention relates to a method for preparing an autologous skin cell agent for alleviating or treating skin defects. The method for preparing a therapeutic agent for alleviating or treating a skin defect using autologous skin cells according to the present invention, comprises the following steps: obtaining normal skin tissue from the patient's body; mincing the obtained tissue into cell units; and obtaining from the minced tissue matters, a skin cell line including keratinocytes, melanocytes and fibroblasts. The present invention excludes an enzyme-treating and/or an incubation step unlike conventional methods for preparing a therapeutic agent, therefore enabling a considerably easier and faster preparation and quicker application to an affected area.
Description
Technical field
The present invention relates to a kind of preparation method for alleviating or treat the autologous skin cellular therapeutic agent of skin injury, relate more specifically to a kind of method for preparing at short notice cellular therapeutic agent because getting rid of enzyme or incubation step that is different from existing cellular therapeutic agent preparation method.
Background technology
Occur in the damaged situation such as burn, wound, depigmentation at skin histology, as suitably treating or take wrong emergency measure, will give the skin scar or cause treatment time to prolong, also may follow other dermatosis simultaneously.
Usually, in burn or the situation of wound, adopted in the past to soak that main component is arranged is that the mode of binder parcel skin of the ointment of vaseline is treated, in order to prevent superinfection, additionally took the biocides such as penicillin, sulfonamide agent.And, in order to alleviate pain disease, take separately analgesics.But in short then 2～3 weeks of Wound healing and bone regeneration required time, long then need long treatment about 3 months, the expense that needs thus is also very high.
And, in the situation of burn, the method that adopts a kind of cellular therapeutic agent of utilizing autogenous cell to make to treat more.But according to the preparation method of utilizing the cellular therapeutic agent that autogenous cell makes in the past, adopt and carry out enzyme and process to extract specific cells, the method that cell culture a few days of said extracted is produced.Therefore, there is the long problem of cell culture required time in cellular therapeutic agent in the past, is not suitable for the patient that burn waits the serious situation of skin injury degree and needs emergency treatment.
Summary of the invention
Technical problem
Therefore, the object of the present invention is to provide a kind of utilization not process the autologous skin cell separate and not cultivate by enzyme comes for the preparation of alleviating or the method for the cellular therapeutic agent for the treatment of skin injury and according to the device of cellular therapeutic agent and the preparation cellular therapeutic agent of said method preparation.
Technical scheme
Above-mentioned purpose can of the present inventionly the method is characterized in that for alleviating or the preparation method of the cellular therapeutic agent for the treatment of skin injury is reached by following, comprise: the step of obtaining normal skin tissue from a donor site that comes from the autologous skin tissue; The above-mentioned normal skin tissue that obtains is ground into the step of cell unit; And from the fragment of tissue that above-mentioned pulverizing forms, obtain and comprise in keratinocyte, melanocyte and the fibrocyte at least a cell in interior Skin Cell group's step.
Wherein, above-mentioned the above-mentioned normal skin tissue that obtains is ground in the step of cell unit, can the using-system destructor, with every 1cm of above-mentioned normal skin tissue size
2Pulverize 10～60 seconds time, thereby above-mentioned normal skin tissue is ground into cell unit.
Wherein, above-mentioned the above-mentioned normal skin tissue that obtains is ground in the step of cell unit, can pulverizes above-mentioned normal skin tissue so that the cell concentration of above-mentioned pulverizing is 1～40 * 10
7Individual/cm
2
Wherein, obtain in the step of normal skin tissue from a donor site that comes from the autologous skin tissue above-mentioned, the above-mentioned keratinocyte that obtains can be always to obtain 60～90% of cell.
Wherein, obtain in the step of normal skin tissue from a donor site that comes from the autologous skin tissue above-mentioned, the above-mentioned melanocyte that obtains can be always to obtain 1～5% of cell.
Wherein, obtain in the step of normal skin tissue from a donor site that comes from the autologous skin tissue above-mentioned, the above-mentioned fibrocyte that obtains can be for always obtaining 10～40% of cell.
Wherein, obtain in the step of normal skin tissue from a donor site that comes from the autologous skin tissue above-mentioned, need not above-mentioned comprise keratinocyte, melanocyte and the fibrocyte that obtains cultivated interior Skin Cell group.
Wherein, obtain in the step of normal skin tissue from a donor site that comes from the autologous skin tissue above-mentioned, be used for obtaining comprising at least a cell of above-mentioned keratinocyte, melanocyte and fibrocyte when interior Skin Cell group, need not to carry out any enzyme and process.
Wherein, above-mentioned skin injury can be select from the group that the skin wound by burn, wound, skin scar, depigmentation consists of a certain.
And purpose of the present invention can be reached by cellular therapeutic agent of the present invention, and this cellular therapeutic agent is prepared from by the preparation method of the cellular therapeutic agent of above-mentioned record.
Beneficial effect
As mentioned above, the preparation method that is used for alleviating or treating the cellular therapeutic agent of skin injury of utilizing the autologous skin cell of the present invention, for the autologous skin cell, need not to adopt the mode of processing to carry out cell separation with enzyme, and isolated cell is not cultivated, can be prepared the effect that cellular therapeutic agent also is used for affected part easily and quickly yet.And, according to the present invention, though few from the amount of the normal skin tissue that main body is obtained, also can be for the preparation of the cellular therapeutic agent of larger area affected part.And, also have the effect that the preparation facilities that can prepare above-mentioned cellular therapeutic agent is provided.
Description of drawings
Fig. 1 is the flow chart of the preparation method of the cellular therapeutic agent of expression one embodiment of the invention.
Fig. 2 is the figure of the example of expression blood cell calculator.
Fig. 3 a to 3b confirms result's figure according to the cell size of the cellular therapeutic agent of one embodiment of the invention preparation for expression.
Fig. 4 for expression according to the cellular therapeutic agent of one embodiment of the invention preparations figure to the clinical effectiveness of burned part.
Fig. 5 for expression according to the cellular therapeutic agent of one embodiment of the invention preparations again figure of a clinical effectiveness to burned part.
Fig. 6 a confirms result's figure for the ultramicroscope of the expression basis isolated cell of enzyme processing mode in the past.
Fig. 6 b confirms result's figure according to the ultramicroscope of the isolated cell of one embodiment of the invention for expression.
Fig. 7 for expression according to the cellular therapeutic agent of one embodiment of the invention preparations figure to the zoopery effect of burned part.
Fig. 8 a is the H﹠amp of the burned part of the above-mentioned Fig. 7 of expression; The figure of E coloration result.
Fig. 8 b is the figure of the MT coloration result of the burned part of the above-mentioned Fig. 7 of expression.
Fig. 9 for expression according to the cellular therapeutic agent of one embodiment of the invention preparations figure to the zoopery effect of wound site.
Figure 10 a is for representing the figure to the MT coloration result behind the applicable matched group of the wound site of Fig. 9.
Figure 10 b is for representing the applicable figure according to the MT coloration result after the cellular therapeutic agent of one embodiment of the invention preparation of the wound site of Fig. 9.
The specific embodiment
Below, with reference to the accompanying drawings embodiments of the invention are elaborated, so that the general technical staff of the technical field of the invention can implement easily.The present invention can realize various embodiment, the embodiment that is not limited in this explanation.
Fig. 1 is the flow chart of the preparation method of cellular therapeutic agent of the present invention.
As shown in Figure 1, the preparation method of cellular therapeutic agent of the present invention comprises: the step (step S11) of obtaining normal skin tissue from a donor site that comes from the autologous skin tissue; The above-mentioned normal skin tissue that obtains is ground into the step (step S12) of cell unit; And the step (step S13) of from the fragment of tissue that above-mentioned pulverizing forms, obtaining the Skin Cell group of at least a cell that comprises in keratinocyte, melanocyte and the fibrocyte.
The preparation method of cellular therapeutic agent of the present invention is for the preparation of in order to alleviate or treat the cellular therapeutic agent of mammiferous skin injury.Preferably, alleviate or the damaged cellular therapeutic agent for the treatment of human skin for the preparation of being used for.
In the obtaining step of above-mentioned normal skin tissue, can utilize dermatome (Dermatome) to obtain normal skin tissue from main body.Aforementioned body can comprise mammal or the mankind.Preferably, from having the human body of skin injury, certain partial skin obtains normal skin tissue.
Above-mentioned normal skin tissue can obtain according to the size cutting of (1 to 5mm) * (1 to 5mm), number with tissue of above-mentioned size can be obtained by the size of considering affected part, as an example, can obtain 1 to 30 of fragment with above-mentioned size, more preferably obtain 5 to 25 fragments, and then preferably obtain 10 to 25 fragments.
The above-mentioned normal skin tissue that obtains is used after can utilizing normal saline or aquesterilisa to clean.
And, can utilize usual way, after being sterilized, the tissue of above-mentioned cleaning is used.
The pulverising step of above-mentioned normal skin tissue is the step that the above-mentioned normal skin tissue that obtains is ground into cell unit.Above-mentioned the above-mentioned normal skin tissue that obtains is ground in the step of cell unit, utilization can be pulverized the general tissue powder millstone of tissue usually.When pulverizing, above-mentioned tissue considers the above-mentioned amount that needs the tissue of pulverizing, can add normal saline on a small quantity or aquesterilisa is pulverized, preferably can add normal saline or the aquesterilisa of 1～10cc and pulverize, more preferably can add normal saline or the aquesterilisa of 1～5cc and pulverize.
Utilize above-mentioned tissue powder millstone to pulverize when organizing, grinding time can consider the amount of above-mentioned tissue and set, preferably, and every 1cm of above-mentioned tissue size
2Can pulverize 1 to 60 second time, more preferably, every 1cm
2Can pulverize 10 to 60 seconds speed, so that above-mentioned tissue powder is broken into cell unit.Pulverize above-mentioned tissue so that the cell concentration of above-mentioned pulverizing is (1 to 40) * 10
7Individual/cm
2The concentration of above-mentioned cell can be according to the state of age of aforementioned body or tissue and difference.
Above-mentioned Skin Cell group's obtaining step represents, finish above-mentioned pulverizing after, from fragment of tissue, obtain the Skin Cell group who comprises at least a cell in keratinocyte, melanocyte and the fibrocyte.
Above-mentioned keratinocyte (keratinocyte) is; produce cutin and pass through to form physical barriers; to play the epidermis cell of the effect of protection individuality from the injurious factor that the outside enters, it is the important structure key element that a kind of generation and the various biological respinse modifier matter of secreting the immunity-regulating reaction are regulated immunoreation and inflammatory reaction.
Above-mentioned melanocyte (melanocyte) is to generate as being present in brown in animal tissue and the skin or the melanic cell of black pigment, and the melanin granule that will generate in cell constantly is supplied to epidermis cell.When the melanin amount was many, skin color was yellowish-brown even pitchy; Measure fewlyer, color relation is more shallow.
Above-mentioned fibrocyte (fibroblast) also is called fibroblast, and it is the cell that consists of the important component of fibrous connective tissue, is flat and rectangular profile when observing by tissue slice, and these cells have irregular projection usually.
Therefore, the above-mentioned step of obtaining normal skin tissue from a donor site that comes from the autologous skin tissue is, obtains the Skin Cell group who comprises keratinocyte, melanocyte and fibrocyte from pulverize the fragment of tissue that above-mentioned tissue forms.
At this moment, above-mentioned comprising of obtaining, at least a cell also may be included in the cell that is destroyed in the pulverising step simultaneously at the cell that interior Skin Cell group not only comprises survival in keratinocyte, melanocyte and the fibrocyte.
Here, the above-mentioned keratinocyte that obtains can account for and always obtain 60～90% of cell, and the above-mentioned melanocyte that obtains can account for and always obtain 1～5% of cell, and the above-mentioned fibrocyte that obtains can account for and always obtain 10～40% of cell.
Therefore, as mentioned above, the application provides a kind of and processes without any enzyme, also can isolate keratinocyte, melanocyte or fibrocellular method from normal skin tissue.And, provide a kind of need not that the above-mentioned at least a cell that comprises in keratinocyte, melanocyte and the fibrocyte that obtains is cultivated interior Skin Cell group, namely can be used for the cellular therapeutic agent of affected part.Thus, the cellular therapeutic agent of the preparation method preparation by the application has not only been got rid of the cell separation process of processing according to enzyme, also got rid of the process of cultivating the cell that separates, can within the very short time, be prepared, can be used for immediately affected part after finishing preparation, thus very effective.
Here, above-mentioned skin injury can be select from the group that the skin wound by burn, wound, skin scar, depigmentation consists of a certain.
Below, will the present invention will be described in more detail by embodiment and experimental example.
The test cell line and the affected part Application Example that comprise in the cellular therapeutic agent of experimental example 1. according to one embodiment of the invention preparation
For 47 years old women and 36 years old women, utilize dermatome, cut respectively 20 normal skin tissues with the size of 4 * 5mm, sterilize after using normal saline to clean.Tissue after the above-mentioned sterilization adds the aquesterilisa of 2cc, and the using-system destructor is pulverized about 30 seconds.
After finishing above-mentioned pulverizing, utilize cell filtration net (cell strainer), from fragment of tissue, obtain and comprise in keratinocyte, melanocyte and the fibrocyte at least a cell interior Skin Cell group, thereby prepare the cellular therapeutic agent of the autologous skin cell that utilizes 47 years old women and utilize the cellular therapeutic agent of 36 years old women's autologous skin cell.
1-1. the concentration determination of cell
Utilize blood cell calculator (Hemocytometer), measure respectively above-mentioned preparation the autologous skin cell that utilizes 47 years old women cellular therapeutic agent and utilize the cell concentration of cellular therapeutic agent of 36 years old women's autologous skin cell.
Fig. 3 represents an embodiment of blood cell calculator.As shown in Figure 2, blood cell calculator is to draw the anyhow object carrier of oblique line on its surface, have two chambers (Chamber) by the regular quadrilateral graduation, the quantity of the cell that can exist in microscopically is determined at the tetragon zoning that is made of prescribed volume of each chamber is determined at the cell concentration that exists in all test portions.
In the cellular therapeutic agent of the 100 μ l that finish above-mentioned preparation, add the trypan blue of 100 μ l, and after evenly mixing, slowly be injected in the blood cell calculator that covers with coverslip.Through after about 1 minute, measure the cell (dead cell) that dyeed and the quantity of undyed cell (survivaling cell) at microscopically.
Measure the concentration of cell by following formula 1.
Formula 1
(1,3,7,9 (Block) sum)
The measurement result of above-mentioned cell concentration shows, utilizes the cellular therapeutic agent of 47 years old women's autologous skin cell, and its cell concentration is every cm
2About 1.4 * 10
8Individual cell; Utilize the cellular therapeutic agent of 36 years old women's autologous skin cell, its cell concentration is every cm
2About 2.4 * 10
8Individual cell.
1-2. cell size is confirmed
Confirm the cellular therapeutic agent of the above-mentioned autologous skin cell that utilizes 47 years old women and utilize keratinocyte, melanocyte and the fibroblastic size that comprises respectively in 36 years old women's the cellular therapeutic agent of autologous skin cell.Step about cell size affirmation experiment adopts common experimental procedure.As a comparative example, confirm former (Primary) cell, i.e. the size of general cell.Above-mentioned cell size is then confirmed by microscope.
The result of above-mentioned cell size experiment is shown in Fig. 3 a to Fig. 3 c.
Fig. 3 a is as above-mentioned comparative example, the result by the microscopic examination archeocyte, Fig. 3 b be by microscopic examination utilize the result of cell size of cellular therapeutic agent of 36 years old women's autologous skin cell, Fig. 3 c be by microscopic examination utilize the result of cell size of cellular therapeutic agent of 47 years old women's autologous skin cell preparation.
Shown in Fig. 3 a, the size of archeocyte (comparative example) is 10-25um; Shown in Fig. 3 b, utilize the cell size that comprises in above-mentioned 36 years old women's the cellular therapeutic agent of autologous skin cell to be 6-15um; Shown in Fig. 3 c, utilize the cell size that comprises in above-mentioned 47 years old women's the cellular therapeutic agent of autologous skin cell to be 6-15um.This shows, utilized above-mentioned 47 years old and the cellular therapeutic agent of 36 years old women's autologous skin cell in the cell size that comprises respectively be in the scope of normal cell size.
1-3. confirm the experiment of cell survival rate
Confirm to utilize above-mentioned 47 years old women the autologous skin cell cellular therapeutic agent and utilize the survival rate of the cell that comprises respectively in 36 years old women's the cellular therapeutic agent of autologous skin cell.In the cellular therapeutic agent of above-mentioned each 100 μ l, add the trypan blue of 100 μ l, and after evenly mixing, slowly be injected in the blood cell calculator that covers with coverslip (with reference to Fig. 2).After about 1 minute, the cell that dyes under the measuring microscope (dead cell) and undyed cell (survivaling cell) quantity utilize following formula 2 to formula 4, calculate cell survival rate.
Total cell number=cell suspension amount of solution (ml) * cell concentration (cell/ml)
Formula 4
Above-mentioned experimental result shows, utilizing the cell survival rate of cellular therapeutic agent of above-mentioned 47 years old women's autologous skin cell is 72%, utilizing the cell survival rate of cellular therapeutic agent of 36 years old women's autologous skin cell is 72.5%, all shows higher cell survival rate.
1-4. affected part Application Example
The cellular therapeutic agent of utilizing above-mentioned 47 years old women's autologous skin cell is used in this 47 years old women's burned part.
At first spray bio-tissue binding agent (fibrin preparation) to above-mentioned burn affected part, and be wrapped with binder after spraying the cellular therapeutic agent of utilizing above-mentioned autologous skin cell.Use above-mentioned cellular therapeutic agent after 7 days, observe its state.
The above results as shown in Figure 4.As shown in Figure 4, the cellular therapeutic agent of preparation in accordance with the present invention preparation is sprayed to the result of burned part, the cell regeneration effect of burned part is remarkable, even can with the naked eye confirm.
Therefore, according to the preparation method of cellular therapeutic agent of the present invention, do not need just can be prepared through steps such as enzyme processing and/or cell culture, quite easy, and can prepare at short notice and be used for affected part, the patient with skin injury for emergency aid and treatment has the effect of highly significant.
For 49 years old male, utilize dermatome, cut 20 normal skin tissues with the size of 3 * 4mm, sterilize after using normal saline to clean.Tissue after the above-mentioned sterilization adds the aquesterilisa of 2cc, and the using-system destructor is pulverized about 30 seconds.
After finishing above-mentioned pulverizing, utilize cell filtration net (cell strainer), from fragment of tissue, obtain and comprise in keratinocyte, melanocyte and the fibrocyte at least a cell interior Skin Cell group, thereby prepare the cellular therapeutic agent of the autologous skin cell that utilizes 49 years old male.
At first spray bio-tissue binding agent (fibrin preparation) to above-mentioned 49 years old male's burned part (cheek and cervical region), be wrapped with binder after spraying the cellular therapeutic agent that contains above-mentioned autologous skin cell.Use above-mentioned cellular therapeutic agent after 10 days, 30 days, 155 days, observe its state.
The result of above-mentioned Application Example as shown in Figure 5.(A) part of Fig. 5 is the photo of male's shooting after just burning in 49 years old.Spray the cellular therapeutic agent that contains above-mentioned autologous skin cell to burned part of above-mentioned Fig. 5 (A) part by the state after 10 days such as (B) part of Fig. 5, sprinkling is by the state after 30 days such as (C) part of Fig. 5, sprays by the state after 100 days such as (D) part of Fig. 5.Hence one can see that, and the cellular therapeutic agent that contains the autologous skin cell of preparation in accordance with the present invention preparation is only sprayed the effect that once just can obtain the regeneration of burned part Skin Cell.Can confirm that from (D) Part of photos taken of Fig. 5 if utilize cellular therapeutic agent of the present invention, the pigment difference on the border between burned skin and the normal skin is few, can expect that the skin pigmentation disorder disease that often occurs because of burn sequelae is had preventive effect.Cellular therapeutic agent of the present invention is very soft, compares the situation of transplanted tissue, can stay the transplanting cicatrix hardly.
Experimental example 3. contains the electron micrograph of the cell of cellular therapeutic agent
The cellular therapeutic agent of preparation in accordance with the present invention preparation comprises the cell that goes out with physical method for separation.To this, by ultramicroscope confirmed the cell that goes out with physical method for separation and in contrast group utilize the isolated cell of enzyme.
Confirm that the result is shown in Fig. 6 a and Fig. 6 b.Fig. 6 a is the electron micrograph that utilizes the isolated cell of enzyme, and Fig. 6 b is the electron micrograph of 49 years old male's of preparation autologous skin cell in above-mentioned experimental example 2.
Usually, cell adheres under the effect of cell adhesion molecule mutually.But shown in Fig. 6 a, process so that in the situation of cell separation, enzyme will cut off above-mentioned cell adhesion molecule by enzyme, can confirm very clearly cell form separately.
Contained cell is not to process to separate by enzyme in the cellular therapeutic agent of the present invention, but realizes separating by physics mode, and therefore, shown in Fig. 6 b, above-mentioned cell adhesion molecule is not disconnected, and still is attached to the cell that separates.So, when cellular therapeutic agent of the present invention is sprayed on affected part, can be attached to well by above-mentioned cell adhesion molecule the tissue of affected part, be conducive to skin regeneration.
Experimental example 4. zooperies
4-1. burned part Application Example
To the effect of burned part, carried out zoopery in order to confirm cellular therapeutic agent prepared in accordance with the present invention.Partial skin at the male nude mouse in 6 ages in week causes burn.By the method identical with the method for above-mentioned experimental example 1 or experimental example 2, the cellular therapeutic agent of utilizing the normal skin tissue of male nude mouse in above-mentioned 6 ages in week to prepare to contain the autologous skin cell is as experimental group.Group is used normal saline in contrast.
Spray 1 matched group and experimental group to the burned part of above-mentioned nude mice at least respectively, after 7 days in burned part is carried out general dressing (dressing) measure, after 14 days above-mentioned nude mice is caused death, and carries out tissue examination.
Making utilizes the piece of tissue of paraffin as the animal tissue of above-mentioned burned part, and is affixed on microscope slide after tissue thinly sliced, to dye.In order to observe cambium and cell etc., above-mentioned microscope slide is carried out hematoxylin-eosin staining (Haematoxylin and eosin stain, H﹠amp; E) observe after and read.And, adopt horse pine trichroism (Masson ' s trichrome) staining to observe and read after collagen protein is dyeed.
Above-mentioned experimental result as shown in Figures 7 and 8.
Fig. 7 is the figure that is illustrated in the Application Example of the cellular therapeutic agent of the present invention on the burned part of nude mice.
As shown in Figure 7, (A) part of Fig. 7 and (C) part of Fig. 7 are the photos of the burned part of the above-mentioned nude mice of expression, Fig. 7 (B) part is to be illustrated in the photo that sprays behind the matched group the 14th day state to the burned part of above-mentioned nude mice, and to be expression spray behind the experimental group photo of the 14th day state to the burned part of above-mentioned nude mice to (D) part of Fig. 7.
In Fig. 7 (B) part, wound location still takes on a red color; In Fig. 7 (D) part, wound location does not take on a red color, but is the color identical with the healthy skin at other positions, and hence one can see that, and wound location skin regeneration ability is remarkable.
Fig. 8 a is expression hematoxylin-eosin staining (Haemato * ylin and eosin stain, H﹠amp; E) result's figure.
Shown in Fig. 8 a, Fig. 8 a (A) part is by normal skin cells is carried out H﹠amp; The photo that M dyeing is obtained is in order to comparative control group and experimental group.Fig. 8 a (B) part is burned part to be used in the situation of matched group carry out H﹠amp; The result of M dyeing can confirm with Normocellular photo and application experiment group and carries out H﹠amp; The result's of M dyeing photo is that (C) part of Fig. 8 a is obviously different.That is, in matched group Fig. 8 a (B) part, almost do not form epithelization (arrow of (B) part of Fig. 8 a), do not carry out the formation of skin accessory organ's (sweat gland, sebaceous gland, hair follicle etc.) fully.On the contrary, in experimental group Fig. 8 a (C) part, almost similarly carried out epithelization with normal structure, skin accessory organ's formation is also very similar to normal cell.
Fig. 8 b is that expression utilizes the as a result figure after horse pine trichrome staining dyes.
Carry out confirming collagen protein the formation degree MT dyeing the result as can be known, be that the immature fibre holostrome is very thin in (A) part of Fig. 8 b at normal cell, and ripe collagen protein (blueness) accounts for the overwhelming majority; In matched group Fig. 8 b (B) part, the non-constant width of immature fibre holostrome d in the wound, and the amount of ripe collagen protein is extremely few; In experimental group Fig. 8 b (C) part, immature fibre holostrome d ' is very thin, and the amount of ripe collagen protein is a lot, and is almost identical with Normocellular amount.
Can confirm according to above-mentioned test, compare matched group, the experimental group of using cellular therapeutic agent of the present invention has significant effect aspect the regeneration of epithelization, skin accessory organ and collagen protein.
4-2. wound site Application Example
To the effect of wound site, carried out zoopery in order to confirm cellular therapeutic agent prepared in accordance with the present invention.Partial skin at the male nude mouse in 6 ages in week causes wound.By the method identical with the method for above-mentioned experimental example 1 or experimental example 2, utilize the normal skin tissue of the male nude mouse in above-mentioned 6 ages in week to prepare the cellular therapeutic agent that contains the autologous skin cell, as test group.Group is used normal saline in contrast.
Spray 1 matched group and test group to the wound site of above-mentioned nude mice at least respectively, after 3 days in wound site is carried out general dressing measure, after 7 days above-mentioned nude mice is caused death, and carries out tissue examination.To above-mentioned wound site, adopt horse pine trichrome staining to the collagen protein rear observation of dyeing, above-mentioned colouring method is identical with method in the above-mentioned 4-1 experimental example.
Fig. 9 is the figure that the wound site that is illustrated in nude mice is used the example of cellular therapeutic agent of the present invention.
As shown in Figure 9, (C) part of Fig. 9 (A) part and Fig. 9 is the photo of the wound site of the above-mentioned nude mice of expression.To be expression spray behind the matched group photo of the 7th day state to the wound site of above-mentioned nude mice to Fig. 9 (B) part, and to be expression spray behind the experimental group photo of the 7th day state to the wound site of above-mentioned nude mice to (D) part of Fig. 9.In the situation of the matched group that uses normal saline, wound location does not form epithelial regeneration fully, and in the situation of the experimental group of the cellular therapeutic agent of the present invention of having used 7 days, wound location is light red, but show the state of epithelial cell regeneration, hence one can see that, and the skin regeneration power of wound location is remarkable.
Figure 10 a uses MT coloration result in the situation of matched group to wound site, and ((A) part of Figure 10 a) after the wound just appearred in expression, use the figure of 200 times of amplifications ((C) part of Figure 10 a) at matched group 7 days ((B) part of Figure 10 a), a position.Shown in Fig. 9 a, in the situation of the matched group that uses normal saline, the epithelization of wound location forms degree low ((B) part of Figure 10 a), the collagen protein very little ((C) part of Figure 10 a) of MT dyeing.
Figure 10 b is to the MT coloration result in the situation of wound site application experiment group, ((A) part of Figure 10 b) after the expression wound, use the figure of 200 times of amplifications ((C) part of Figure 10 b) at matched group 7 days ((B) part of Figure 10 b), b position.
Shown in Figure 10 b, compare matched group, in the experimental group that uses cellular therapeutic agent of the present invention, epithelization forms activity more vigorous ((B) part of Figure 10 b), the amount of collagen protein (blueness) relatively more ((C) parts of Figure 10 b).Therefore, compare matched group, the regeneration capacity of using epithelization in the experimental group of cellular therapeutic agent of the present invention and collagen protein is more remarkable.
Below only illustrate and some embodiment of the present invention have been described, the general technical staff of the technical field of the invention can be out of shape present embodiment in principle of the present invention or thought range.Invention scope is by claim and be equal to alternative the decision.
Claims (10)
1. a preparation method that is used for alleviating or treating the cellular therapeutic agent of skin injury is characterized in that, comprising:
From a donor site that comes from the autologous skin tissue, obtain the step of normal skin tissue;
The above-mentioned normal skin tissue that obtains is ground into the step of cell unit; And
From the fragment of tissue that above-mentioned pulverizing forms, obtain the Skin Cell group's who comprises at least a cell in keratinocyte, melanocyte and the fibrocyte step.
2. the preparation method of cellular therapeutic agent according to claim 1 is characterized in that, above-mentioned the above-mentioned normal skin tissue that obtains is ground in the step of cell unit, and the using-system destructor is with every 1cm of above-mentioned normal skin tissue size
2Pulverize 10～60 seconds time, thereby above-mentioned normal skin tissue is ground into cell unit.
3. the preparation method of cellular therapeutic agent according to claim 1 is characterized in that, above-mentioned the above-mentioned normal skin tissue that obtains is ground in the step of cell unit, pulverizes above-mentioned normal skin tissue so that the cell concentration of above-mentioned pulverizing is 1～40 * 10
7Individual/cm
2
4. the preparation method of cellular therapeutic agent according to claim 1, it is characterized in that, obtain in the step of normal skin tissue from a donor site that comes from the autologous skin tissue above-mentioned, the above-mentioned keratinocyte that obtains is for always obtaining 60～90% of cell.
5. the preparation method of cellular therapeutic agent according to claim 1, it is characterized in that, obtain in the step of normal skin tissue from a donor site that comes from the autologous skin tissue above-mentioned, the above-mentioned melanocyte that obtains is for always obtaining 1～5% of cell.
6. the preparation method of cellular therapeutic agent according to claim 1, it is characterized in that, obtain in the step of normal skin tissue from a donor site that comes from the autologous skin tissue above-mentioned, the above-mentioned fibrocyte that obtains is for always obtaining 10～40% of cell.
7. the preparation method of cellular therapeutic agent according to claim 1, it is characterized in that, obtain in the step of normal skin tissue from a donor site that comes from the autologous skin tissue above-mentioned, need not the above-mentioned at least a cell that comprises in keratinocyte, melanocyte and the fibrocyte that obtains is cultivated interior Skin Cell group.
8. the preparation method of a cellular therapeutic agent, it is characterized in that, obtain in the step of normal skin tissue from a donor site that comes from the autologous skin tissue above-mentioned, be used for obtaining comprising at least a cell of keratinocyte, melanocyte and fibrocyte when interior Skin Cell group, need not to carry out enzyme and process.
9. the preparation method of cellular therapeutic agent according to claim 1 is characterized in that, above-mentioned skin injury is a certain for what select the group who consists of from the skin wound by burn, wound, skin scar, depigmentation.
10. a cellular therapeutic agent is characterized in that, by being prepared from such as each described method in the claim 1 to 9.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2010-0044179 | 2010-05-11 | ||
| KR20100044179 | 2010-05-11 | ||
| PCT/KR2011/003474 WO2011142588A2 (en) | 2010-05-11 | 2011-05-11 | Method for preparing autologous skin cell agent for alleviating or treating skin defects |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103037871A true CN103037871A (en) | 2013-04-10 |
Family
ID=44914822
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011800228845A Pending CN103037871A (en) | 2010-05-11 | 2011-05-11 | Method for preparing autologous skin cell agent for alleviating or treating skin defects |
Country Status (3)
| Country | Link |
|---|---|
| KR (1) | KR101328561B1 (en) |
| CN (1) | CN103037871A (en) |
| WO (1) | WO2011142588A2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611289A (en) * | 2015-01-13 | 2015-05-13 | 广州赛奕德生物技术有限公司 | Method for simultaneously preparing autologous epidermal cells and fibroblasts, and biological beauty product comprising autologous epidermal cells and fibroblasts |
| CN107961061A (en) * | 2017-12-19 | 2018-04-27 | 济南磐升生物技术有限公司 | A kind of autologous skin wound therapeutical devices |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102427018B1 (en) * | 2019-06-11 | 2022-07-29 | 재단법인 베스티안재단 | Method for preparing treating agent for skin and treating agent for skin therefrom |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8394371B2 (en) | 2002-02-11 | 2013-03-12 | Neocutis Sa | Compositions for the treatment of skin conditions, disorders or diseases and methods of making and using the same |
| US7641898B2 (en) * | 2003-03-21 | 2010-01-05 | Materials Evolution And Development Usa, Inc. | Keratinocyte-fibrocyte concomitant grafting for wound healing |
| KR100806695B1 (en) * | 2005-11-25 | 2008-02-27 | 주식회사 엠씨티티 | Pharmaceutical Compositions for Cell Therapy of Pigmentation Disorders |
-
2011
- 2011-05-11 CN CN2011800228845A patent/CN103037871A/en active Pending
- 2011-05-11 KR KR1020110043780A patent/KR101328561B1/en not_active IP Right Cessation
- 2011-05-11 WO PCT/KR2011/003474 patent/WO2011142588A2/en active Application Filing
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611289A (en) * | 2015-01-13 | 2015-05-13 | 广州赛奕德生物技术有限公司 | Method for simultaneously preparing autologous epidermal cells and fibroblasts, and biological beauty product comprising autologous epidermal cells and fibroblasts |
| CN107961061A (en) * | 2017-12-19 | 2018-04-27 | 济南磐升生物技术有限公司 | A kind of autologous skin wound therapeutical devices |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2011142588A9 (en) | 2012-06-07 |
| WO2011142588A3 (en) | 2012-04-19 |
| KR101328561B1 (en) | 2013-11-13 |
| WO2011142588A2 (en) | 2011-11-17 |
| KR20110124722A (en) | 2011-11-17 |
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