JP2024117299A - Method and device for producing composition for tissue regeneration - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition for tissue regeneration which enables less invasive regenerative medicine.

SOLUTION: A method for producing a composition for tissue regeneration according to the present invention comprises: a step for extracting adipose tissue-derived stem cells from a first adipose tissue collected from a human body; a step for culturing the adipose tissue-derived stem cells; and a step for mixing the cultured adipose tissue-derived stem cells with a second adipose tissue newly collected from the human body. The weight of each of the first adipose tissue and the second adipose tissue may be 5-30 g inclusive.

SELECTED DRAWING: Figure 1

COPYRIGHT: (C)2024,JPO&INPIT

Description

The present invention relates to a method and device for producing a tissue regeneration composition for use in regenerative medicine.

Regenerative medicine using bone marrow-derived mesenchymal stem cells (MSCs) has been attracting attention. MSCs are one of the cell groups that make up bone marrow tissue, and have the ability to differentiate into fat cells, bone-forming cells, chondrogenic cells, and muscle-forming cells. As such, it is expected that they will be used for transplantation into the human body, such as for the treatment of pelvic floor dysfunction and for transplantation into the mammary gland after breast cancer surgery.

For example, the pelvic floor muscles are located at the bottom of the pelvis (between the pubic bone, coccyx, and ischium), and when the function of the pelvic floor muscles decreases, pelvic floor dysfunction occurs.

Pelvic floor dysfunction, for example, causes urinary incontinence due to impaired urethral sphincter function. In women, it can be caused by weakening of the pelvic floor muscles due to pregnancy, childbirth, or aging, or by sphincter dysfunction due to gynecological surgery. In men, it can be caused by sphincter dysfunction due to benign prostatic hyperplasia or prostate cancer surgery.

Fecal incontinence is also caused by a weakening of the contractile strength of the external and internal anal sphincter muscles. In addition to muscle weakness associated with aging, a weakening of the external anal sphincter function can also be caused in women by anal sphincter rupture due to perineal tears during vaginal childbirth.

Although the decline in function of the internal anal sphincter often occurs with age, it can also result from damage to the internal anal sphincter caused by perineal tears following vaginal childbirth or after rectal surgery such as rectal resection.

These pelvic floor dysfunctions interfere with many areas of daily life and significantly impair quality of life (QOL), so there is a demand for minimally invasive, highly effective treatments.

However, in regenerative medicine using MSCs, collecting autologous bone marrow tissue as MSCs places a heavy burden on the patient and poses problems such as the limited number of cells that can be collected.

Therefore, as an alternative cell source to bone marrow tissue, there is a need for tissues and cells that can be collected in large quantities under local anesthesia in a way that places minimal strain on the patient.

The possibility of using stem cells collected from human adipose tissue as an alternative cell source has been shown. Processed lipoaspirate (PLA) cells, obtained by isolating adipose tissue aspirated by liposuction, differentiate into adipogenic cells, chondrogenic cells, myogenic cells, and osteogenic cells under culture conditions in the presence of specific differentiation factors.

It has also been reported that adipose tissue-derived stem cells (ASCs) contribute to adipose tissue engraftment by promoting angiogenesis (Non-Patent Document 1).

As described above, adipose tissue-derived stem cells (ASCs) present in subcutaneous adipose tissue are promising stem cells for use in regenerative medicine because they can be easily harvested and transplanted in large numbers and are highly safe compared to bone marrow-derived stem cells.

Zhu M, et al.Supplementation of fat grafts with adipose-derived regenerative cells improves long-term graft retention. Plast Surg. 2010;64(2):222-8.

However, the large amount of adipose tissue required for ASC (approximately 300 g) must be harvested under general anesthesia, making it a highly invasive treatment that places a heavy burden on patients.

In order to solve the problems described above, the method for producing a composition for tissue regeneration according to the present invention comprises the steps of extracting adipose tissue-derived stem cells from a first adipose tissue harvested from a human body, culturing the adipose tissue-derived stem cells, and mixing the cultured adipose tissue-derived stem cells with a second adipose tissue freshly harvested from the human body.

In addition, in the method for producing a composition for tissue regeneration according to the present invention, the weight of the first adipose tissue and the second adipose tissue may be 5 grams or more and 30 grams or less.

In addition, in the method for producing a tissue regeneration composition according to the present invention, the second adipose tissue may be collected using an 18G capillary tube.

The method for producing a tissue regeneration composition according to the present invention may also be such that the tissue regeneration composition is injected around the anus.

The tissue regeneration composition manufacturing apparatus according to the present invention further comprises a stem cell introduction section into which adipose tissue-derived stem cells generated from a first adipose tissue collected from a human body are introduced, an adipose tissue introduction section into which a second adipose tissue newly collected from the human body is introduced, and a stirring section that stirs the adipose tissue-derived stem cells introduced from the stem cell introduction section and the adipose tissue introduced from the adipose tissue introduction section.

The apparatus for producing a tissue regeneration composition according to the present invention may also include an extraction unit that extracts the adipose tissue-derived stem cells from the first adipose tissue, and a culture unit that cultures the adipose tissue-derived stem cells introduced from the extraction unit.

The tissue regeneration composition manufacturing device according to the present invention may also include a first weight measuring unit connected to the stem cell introduction unit and a second weight measuring unit connected to the adipose tissue introduction unit.

The present invention provides a method and device for producing a tissue regeneration composition that enables minimally invasive regenerative medicine.

FIG. 1 is a flow chart for explaining a method for producing a composition for tissue regeneration according to a first embodiment of the present invention. FIG. 2 is a block diagram showing the configuration of an apparatus for producing a composition for tissue regeneration according to the first embodiment of the present invention. FIG. 3 is a diagram for explaining the effect of the method for producing a composition for tissue regeneration according to the first embodiment of the present invention.

First Embodiment
An example of a method for producing the composition for tissue regeneration according to the first embodiment of the present invention will be described with reference to FIG.

<Method of producing a composition for tissue regeneration>
An example of a method for producing a composition for tissue regeneration according to this embodiment will be described with reference to FIG.

First, adipose tissue (first adipose tissue) is collected from a human body (patient, subject). For example, after making a small incision of about 3 mm in the lower abdomen (or thigh, abdomen, etc.), about 10 g of adipose tissue is collected using a syringe equipped with an 18G injection needle. Alternatively, several skin incisions of about 1 cm are made in the thigh or abdomen, which are the suction sites, and the adipose tissue is aspirated using a dedicated tube (suction cannula). Alternatively, a skin incision of about 1 cm is made in the abdomen or thigh, and subcutaneous fat is collected. Here, it is desirable that the amount of collected adipose tissue is 5 g or more. It is even more desirable that it is 30 g or less.

First, adipose tissue-derived stem cells are extracted from the collected adipose tissue (first adipose tissue) using a cell separation device (e.g., the "Celution Centrifuge" manufactured by Cytori Therapeutics, Inc.) (step S1). Here, about 0.2 g of adipose tissue-derived stem cells are extracted. In the extraction process, enzymes such as collagenase, trypsin, or dispase are used alone or in combination to separate the adipose tissue-derived stem cells. Here, all or a portion of the collected adipose tissue may be used.

Next, the extracted adipose tissue-derived stem cells (about 0.2 g) are cultured to increase the volume to about 5.0 mL (step S2). Here, the culture can be performed by a general method. For example, a medium is added to the extracted adipose tissue-derived stem cells, a cell suspension is prepared so that the cell concentration is 4 x 10 ⁴ cells/cm ² , and the cell suspension is seeded in a culture vessel. After seeding, the cells are cultured in an incubator at 37°C and 5% CO _2. Here, the cell concentration is measured by aspirating the cells with a syringe and visually checking the scale of the syringe. In addition, the medium may be a medium generally used for culturing adipose tissue-derived stem cells, such as a medium for human embryonic stem cells or a medium for culturing adipose precursor cells.

Next, approximately 10 g of new adipose tissue (second adipose tissue) is collected from the same body (patient, test subject) from which the first adipose tissue was collected, by suction with a syringe equipped with an 18G injection needle. For example, after making a small incision of approximately 3 mm in the lower abdomen (or thigh, abdomen, etc.), approximately 10 g of adipose tissue is collected with a syringe equipped with an 18G injection needle. Here, it is desirable that the amount of collected adipose tissue be 5 g or more. Furthermore, it is desirable that the amount be 30 g or less.

Here, it is desirable to use an injection needle or cannula of about 18G (inner diameter 0.90 mm) to collect new adipose tissue (second adipose tissue). If a thin injection needle is used to collect the second adipose tissue, it is difficult to aspirate the adipose tissue. Furthermore, if a thick injection needle is used, the collected adipose tissue is too large to pass through an injection needle of about 18G that is normally used for injection. As a result, it is difficult to inject the adipose tissue collected with a thick injection needle into the target area of the human body.

In addition, because adipose tissue is hard and fibrous, it is difficult to break down large pieces of tissue after harvesting them.

The collected adipose tissue (second adipose tissue) in this syringe is washed with lactated Ringer's solution, and the amount of adipose tissue is then adjusted to 8.0 mL.

Finally, the cultured adipose tissue-derived stem cells (1.6-2.0 mL) are injected into the 8.0 mL of adipose tissue (second adipose tissue) in the syringe, and stirred and mixed to produce a mixture of adipose tissue-derived stem cells and adipose tissue, i.e., a composition for tissue regeneration (about 10 mL) (step S3). At this time, the mixture is stirred gently and sufficiently as soon as possible so as not to apply pressure to the cells. Here, the adipose tissue-derived stem cells are about 1 x 10 ⁶ -2 x 10 ⁸ cells, the adipose tissue is about 1-300 mL, and the temperature is about 4-10°C. The amount of cells and the amount of adipose tissue are measured by aspirating the cells and the adipose tissue with a syringe and visually checking the scale on the syringe.

In this way, the mixture of adipose tissue-derived stem cells and adipose tissue, i.e., the tissue regeneration composition, is produced by placing it in a syringe equipped with the 18G injection needle used to harvest the new adipose tissue (second adipose tissue).

As described above, the tissue regeneration composition according to this embodiment is manufactured.

<Method of Transplanting Tissue Regeneration Composition>
The tissue regeneration composition of this embodiment, manufactured as described above, is injected (administered) around the target site (e.g., urethra or anus) using the syringe equipped with the 18G injection needle that was used to mix the adipose tissue-derived stem cells and adipose tissue.

For example, to improve urinary incontinence, the tissue regeneration composition is injected submucosally into the urethral sphincter at two or several locations. To improve fecal incontinence, the tissue regeneration composition is injected into the external anal sphincter or internal anal sphincter at two or several locations. In this manner, the tissue regeneration composition is transplanted.

Here, to inject the tissue regeneration composition into the submucosa of the urethral sphincter and into minute locations in the external or internal anal sphincter, an injection needle or cannula of about 18 G (inner diameter 0.90 mm) or a needle or cannula smaller than 18 G, for example 19 G (inner diameter 0.70 mm), is used.

In this way, the syringe equipped with the 18G needle used to harvest the new adipose tissue is used to inject (administer) the tissue into the target area, ensuring cleanliness and preventing the newly harvested adipose tissue from coming into contact with the outside air.

In this embodiment, an example has been shown in which adipose tissue-derived stem cells and adipose tissue are mixed and injected (administered) as a tissue regeneration composition around the target site, but this is not limiting. Adipose tissue-derived stem cells and adipose tissue may each be injected around the target site. In this case, adipose tissue-derived stem cells may be injected after injection (administration). Adipose tissue may be injected after injection of adipose tissue-derived stem cells. Alternatively, only adipose tissue-derived stem cells may be injected, or only adipose tissue may be injected.

<Apparatus for producing composition for tissue regeneration>
An example of an apparatus for producing a composition for tissue regeneration according to this embodiment will be described with reference to FIG.

As shown in FIG. 2, the tissue regeneration composition manufacturing device 10 includes a first adipose tissue introduction section 11, an extraction section 12, a culture section 13, and a composition production section 14.

Adipose tissue (first adipose tissue) collected from a human body (patient, subject) is introduced into the first adipose tissue introduction section 11.

The extraction unit 12 has a configuration similar to that of a normal cell separation device, and extracts adipose tissue-derived stem cells from the adipose tissue introduced into the first adipose tissue introduction unit 11.

The culture unit 13 includes a culture chamber 131, a gas introduction unit 132, and a culture temperature control unit 133, and cultures the extracted adipose tissue-derived stem cells. For example, a cell suspension prepared by adding a preadipocyte culture medium to adipose tissue-derived stem cells is seeded in a culture container in the culture chamber 131. The gas introduction unit 132 mixes the gas with _CO2 , and 5% _CO2 gas is introduced into the culture chamber 131. The culture temperature control unit 133 controls the temperature in the culture chamber 131 to a predetermined temperature (e.g., 37°C).

The composition generating unit 14 includes a stem cell introduction unit 141, a second adipose tissue introduction unit 142, weight measuring units 143 and 144 connected to the stem cell introduction unit 141 and the second adipose tissue introduction unit 142, a stirring unit 145, a stirring control unit 146 and a stirring temperature control unit 147 connected to the stirring unit, a collection unit 148, and a cell delivery tube 149.

In the composition generating section 14, cultured adipose tissue-derived stem cells are introduced into the stem cell introduction section 141.

Newly harvested adipose tissue (second adipose tissue) from the same human body (patient, subject) from which the first adipose tissue was harvested is introduced into the second adipose tissue introduction section 142.

The weight measuring unit 143 measures the weight of the introduced stem cells. When the weight of the introduced stem cells is a predetermined weight, for example, about 1 to 10 g, the stem cells are sent to the agitation unit 145.

The weight measuring unit 144 measures the weight of the introduced adipose tissue. When the weight of the introduced adipose tissue is a predetermined weight, for example, about 5 to 30 g, the adipose tissue is sent to the agitation unit 145.

The stirring unit 145 stirs and mixes the stem cells and the adipose tissue. Here, the stirring control unit 146 controls the rotation or vibration during stirring, and the stirring temperature control unit 147 controls the temperature during stirring.

The tissue regeneration composition produced is discharged and collected in the collection section 148.

At this time, the collected tissue regeneration composition may be stored in a syringe or the like to be used for injection in a space that is not exposed to the outside air (for example, a space evacuated by a vacuum pump or a space filled with an inert gas). This ensures cleanliness and allows the tissue regeneration composition to be stored in a syringe or the like to be used for injection.

The cell delivery pipe 149 connects the stem cell introduction section 141, the second adipose tissue introduction section 142, the weight measurement sections 143 and 144 connected to each other, the stirring section 145, and the collection section 148, and the stem cells, adipose tissue, and tissue regeneration composition are delivered via the cell delivery pipe 149. A pump (not shown) may be used to deliver the stem cells, adipose tissue, and tissue regeneration composition.

In addition, the transfer of adipose tissue-derived stem cells from the extraction unit 12 to the culture unit 13 may be automatic or manual.

In addition, the introduction of adipose tissue-derived stem cells into the stem cell introduction section 141 may be automatic or manual.

The tissue regeneration composition manufacturing device according to this embodiment may be composed of only a composition production section, or adipose tissue-derived stem cells that have been extracted and cultured in advance and freshly collected adipose tissue may be introduced into the stem cell introduction section and the second adipose tissue introduction section, respectively.

In addition, a weight measuring unit does not have to be provided, and the weight may be measured and adjusted in advance before introducing the adipose tissue-derived stem cells and adipose tissue.

As described above, the tissue regeneration composition produced by the manufacturing device according to this embodiment is injected into the area surrounding the target site (e.g., the urethra or anus), and the tissue regeneration composition is transplanted.

＜Effects＞
The effects of the tissue regeneration composition according to the first embodiment of the present invention will be described.

First, the effect of adipose tissue-derived stem cells (ASCs) on urinary incontinence is described below. In detail, adipose tissue-derived stem cells (ASCs) were transplanted around the urethral sphincter of lysyloxidase-like-1 knockout (LOXL1-KO) rats, which have inhibited polymerization of elastic fibers, as a model of stress urinary incontinence, and their urinary continence effect was evaluated.

ASCs were extracted from the subcutaneous fat tissue in the inguinal region of 5-week-old SD rats and expanded at 37°C in a 5% _CO2 environment. They were then labeled with PKH26 and 1.0 × ¹⁰⁶ labeled ASCs were transplanted around the urethra of LOXL1-KO rats (transplant group).

For comparison, PBS was administered to SD rats as a control group and evaluated in the same manner (PBS-administered group). SD rats were also administered the sham operation group and evaluated in the same manner.

Five mice from each group were used to measure abdominal leak point pressure (ALPP) two and four weeks after transplantation, and tissues were excised and subjected to histological observation.

As a result, ALPP in the control group was significantly lower than in the sham-operated group both 2 and 4 weeks after transplantation (p<0.01). Furthermore, ALPP in the transplanted group was at the same level as in the sham-operated group, and was significantly higher than in the control group (p<0.01). No significant changes in ALPP were observed in either group 2 and 4 weeks after transplantation.

Furthermore, hematoxylin and eosin (HE) staining did not reveal any significant thickening of the urethral wall or narrowing of the lumen in the transplant group. On the other hand, immunostaining revealed double positive cells for PKH26 and α-SMA.

In this way, transplanting ASCs around the urethra can improve the amount of urine leakage and is effective against stress urinary incontinence.

This effect may also be due to a physical blockage of the urethra, or alternatively, transplanted ASCs may partially differentiate into smooth muscle, increasing urethral resistance.

Next, the effect of the tissue regeneration composition according to this embodiment on fecal incontinence will be described below.

In particular, as a basic experiment to verify the effect of the tissue regeneration composition according to the present embodiment, adipose tissue-derived stem cells (ASCs) and adipose tissue were injected into the anal area of a rat model with mechanical anal sphincter injury, and the fecal continence effect was evaluated. Here, injecting ASCs and adipose tissue into the anal area has the same effect as transplanting the tissue regeneration composition according to the present embodiment into the anal area.

ASCs were extracted from subcutaneous adipose tissue in the inguinal region of 5-week-old SD rats and expanded at 37°C in a 5% _CO2 environment, after which 0.1 mL of ASCs and 0.1 mL of adipose tissue were injected once into the anal region (four sites at 3, 6, 9, and 12 o'clock) of a rat model with mechanical anal sphincter injury (ASCF-S group). In addition, injections (transplants) were performed twice in the same manner (ASCF-D group).

For comparison, phosphate-buffered saline (PBS) was injected in a similar manner (PBS group). Similarly, 0.1 mL of ASC was injected once (ASC-S group) or twice (ASC-D group).

Two weeks after transplantation, five animals from each group were used to measure the anal pressure profile (APP) as follows. An anal pressure profile recording catheter with a diameter of 1.5 mm, a blind tip, and a side hole with a diameter of 1 mm 6 mm from the tip was used. This catheter was inserted into the rectum from the anus under urethane anesthesia. The catheter was then fixed to the outer barrel of a syringe installed in a syringe pump. Next, while continuously injecting saline into the catheter at 12 mL/h, the catheter was withdrawn at the speed of the syringe pump (approximately 25 mm/min) and the APP was measured.

Figure 3 shows the APP results for each group two weeks after transplantation. For comparison, the measurement results for untreated SD rats are also shown.

The intraanal pressure in untreated rats is about 11.5 cmH ₂ O. The intraanal pressure in the PBS group is 4.0 cmH ₂ O, and in the ASC-S and ASC-D groups is about 4.0-4.5 cmH ₂ O. Thus, when ASC alone is injected, no increase in intraanal pressure is observed.

On the other hand, the intraanal pressure was about 5.0 cmH ₂ O in the ASCF-S group, but increased to 8.0 cmH ₂ O or more in the ASCF-D group. Thus, an increase in intraanal pressure was observed by injecting ASCs and fat cells.

Therefore, fecal incontinence can be suppressed by injecting adipose tissue-derived stem cells (ASCs) and adipose tissue into the anal area. This shows that the tissue regeneration composition according to the present embodiment has a fecal incontinence effect.

The tissue regeneration composition according to this embodiment has the following effects:

1) The tissue regeneration composition of the present invention functions as a scaffold for engraftment, allowing adipose tissue-derived stem cells to differentiate into smooth muscle, external anal sphincter, and internal anal sphincter and engraft without being degraded or absorbed in the body, thereby improving the engraftment rate.

2) Similarly, the tissue regeneration composition of the present invention can be used as a scaffold to improve the survival rate of adipose tissue.

3) The adipose tissue contained in the tissue regeneration composition of the present invention can increase the volume of autologous tissue at the cell transplantation site (bulking effect).

4) The transplanted adipose tissue-derived stem cells differentiate and engraft into smooth muscle, the external anal sphincter, and the internal anal sphincter, and promote tissue proliferation in the surrounding tissues, thereby increasing muscle contraction pressure.

The tissue regeneration composition according to this embodiment allows for a reduction in the amount of adipose tissue harvested from the human body, making it possible to achieve minimally invasive treatment in which a small amount (5-10 g) of adipose tissue is harvested under local anesthesia. As a result, the burden on the patient can be reduced, and pelvic floor dysfunction such as urinary incontinence and fecal incontinence can be improved.

In addition, in conventional transplantation methods using tissue regeneration compositions, the tissue regeneration composition can be administered only once. On the other hand, in the method for producing the tissue regeneration composition according to the present embodiment, cells can be multiplied by culturing, so that cells for multiple administrations can be secured. Therefore, cells for the second and subsequent administrations can be banked and stored, and used (administered) as needed. Thus, according to the tissue regeneration composition according to the present embodiment, the tissue regeneration composition can be administered multiple times during transplantation.

In the embodiment of the present invention, an example is shown in which a syringe needle is used to collect adipose tissue, etc., but this is not limited to this, and thin tubes such as cannulas or needles used in other medical devices may also be used.

In the embodiment of the present invention, the tissue regeneration composition is applied to transplantation around the urethra and around the anus, but it may also be applied to other pelvic floor muscles, mammary glands after breast cancer surgery, gums, paralyzed vocal cords, joints, spine, etc.

In the embodiment of the present invention, examples of the structure, dimensions, materials, etc. of each component part in the configuration of the method and device for producing a tissue regeneration composition are shown, but the present invention is not limited to these. Any method and device for producing a tissue regeneration composition may be used as long as they exhibit the functions and effects.

The present invention relates to a method and device for producing a tissue regeneration composition, and can be applied to tissue regeneration compositions and medical devices used for transplantation to suppress urinary incontinence and fecal incontinence.

10: Apparatus for producing a composition for tissue regeneration 11: First adipose tissue introduction section 12: Extraction section 13: Culture section 131: Culture chamber 132: Gas introduction section 133: Culture temperature control section 14: Composition production section 141: Stem cell introduction section 142: Second adipose tissue introduction section 143, 144: Weight measurement section 145: Stirring section 146: Stirring control section 147: Stirring temperature control section 148: Recovery section 149: Cell delivery pipe

Claims (7)

Extracting adipose tissue-derived stem cells from a first adipose tissue harvested from a human body;
Culturing the adipose tissue-derived stem cells;
mixing the cultured adipose tissue-derived stem cells with a second adipose tissue that is freshly harvested from the human body. The method for producing a composition for tissue regeneration according to claim 1, characterized in that the weight of the first adipose tissue and the second adipose tissue is 5 grams or more and 30 grams or less. The method for producing a composition for tissue regeneration according to claim 1 or 2, characterized in that the second adipose tissue is collected using an 18G capillary tube. The method for producing a composition for tissue regeneration according to claim 1 or 2, wherein the composition for tissue regeneration is a composition for treating fecal incontinence. a stem cell introduction section into which adipose tissue-derived stem cells generated from a first adipose tissue collected from a human body are introduced;
An adipose tissue introduction section into which second adipose tissue newly harvested from the human body is introduced;
an agitation section that agitates and mixes the adipose tissue-derived stem cells introduced through the stem cell introduction section and the adipose tissue introduced through the adipose tissue introduction section. an extraction unit that extracts the adipose tissue-derived stem cells from the first adipose tissue;
The apparatus for producing a composition for tissue regeneration according to claim 5 , further comprising: a culture section for culturing the adipose tissue-derived stem cells introduced from the extraction section. A first weight measuring unit connected to the stem cell introduction unit;
The apparatus for producing a composition for tissue regeneration according to claim 5 or claim 6, further comprising: a second weight measuring section connected to the adipose tissue introduction section.

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