The present invention primarily relates to a device for constructing a three-dimensional muscle tissue and a method for producing a three-dimensional muscle tissue.
There is an increasing demand for edible meat due to population growth and rising incomes in emerging countries. However, increasing the meat supply is difficult due to soaring prices of grains for livestock feed and problems in securing breeding places. The development of meat alternatives has thus been hoped for.
Cultured meat is produced by forming tissue using skeletal muscle cells proliferated in culture. Since production is possible in a laboratory, production that is not affected by climate change is possible. Additionally, greenhouse gas emissions are low, and the environmental impact is small, compared to conventional animal husbandry.
For producing edible meat with a steak-like texture from cells, construction of cultured meat in which the direction of muscle fibers is well oriented (referred to below as “three-dimensional muscle tissue”) is necessary. However, conventional techniques only provide minced meat, in which the direction of muscle fibers is not well oriented. Although three-dimensional muscle tissues obtained using human or mouse skeletal muscle cells have been reported in the field of regenerative medicine, the tissues do not have sufficient thickness (Patent Literature (PTL) 1 and PTL 2).
An object of the present invention is to provide a device for constructing a three-dimensional muscle tissue for producing a three-dimensional muscle tissue, and a method for producing a three-dimensional muscle tissue.
The present invention provides the following device for constructing a three-dimensional muscle tissue and method for producing a three-dimensional muscle tissue.
[1]
A device for constructing a three-dimensional muscle tissue, comprising
[2]
The device for constructing a three-dimensional muscle tissue according to Item [1], further comprising channel-forming members for culture medium that are engaged to the first support members and the second support members.
[3]
The device for constructing a three-dimensional muscle tissue according to Item [1] or [2], further comprising a pump for supplying culture medium to the culture medium inlet.
[4]
A method for producing a three-dimensional muscle tissue, the method comprising, in the device for constructing a three-dimensional muscle tissue of any one of Items [1] to [3],
[5]
A three-dimensional muscle tissue comprising
[6]
The three-dimensional muscle tissue according to Item [5], wherein the interval between adjacent channels is 500 to 1500 μm.
[7]
The three-dimensional muscle tissue according to Item [5] or [6], wherein the direction of orientation of the channels and the direction of orientation of the muscle fibers are approximately parallel.
[8]
The three-dimensional muscle tissue according to any one of Items [5] to [7], which does not substantially comprise heme.
The present invention provides, in particular, a device for constructing a three-dimensional muscle tissue and a production method. The device etc. of the present invention enable production of three-dimensional muscle tissue with sufficient thickness. Thus, the three-dimensional muscle tissue produced according to the present invention can be expected to have a steak-like texture. Additionally, in regenerative medicine applications, damage in extensive and thick muscle tissue is expected to be healed with a single treatment by using thick three-dimensional muscle tissue.
The device 1 for constructing a three-dimensional muscle tissue of the present invention comprises a first connector 4 and a second connector 5, which are provided opposite to each other, and channels 3 for culture medium, which are foiled between first support members 8 and second support members 9.
In one embodiment of the present invention, the channels 3 can be famed by filling a culture space 2 between the first connector and the second connector with a hydrogel containing skeletal myoblasts, with solid channel-forming members such as wire or acupuncture needles, being inserted between the first support members 8 and the second support members 9 (
In
The culture medium 15 is supplied by a pump 12 to a culture medium inlet 6 and allowed to flow from the first support members 8 through the channels 3 to the second support members 9. The culture medium 15 containing waste after passing through the second support members 9 is discharged from a culture medium outlet 7 famed at the second connector 5. Each of the first support members 8 and the second support members 9 has an opening into which the channel-forming member 10 is inserted. The sealing material 20 is for sealing the openings on the side of the first support members. The sealing material 20 allows the culture medium 15 introduced from the culture medium inlet 6 to flow through the channels 3 in the direction of the arrows in
The surface of the cultured product of the skeletal myoblasts 13 (three-dimensional muscle tissue 16) is well supplied with the nutrients etc. 14 from the culture medium 15, the interior of the muscle tissue 16 is supplied with the nutrients etc. 14 from a fresh culture medium flowing through the channels 3, and a waste liquid with high concentrations of carbon dioxide and waste is discharged from the culture medium outlet 7. In
The first support members and the second support members are preferably disposed in a straight line so as to form straight channels. The interval between adjacent channels depend on the thickness (diameter or cross-sectional area) of the channels, but is preferably about 500 to 1500 μm.
As shown in the cross-sectional views in
The first and second muscle tissue anchors 11 may have any shape, and preferably have a mesh-like shape as shown in
Electrical stimulation may be applied during culture of the muscle tissue 16 to promote proliferation of the skeletal myoblasts 13.
In one preferable embodiment of the present invention, the oxygen concentration in the culture medium is preferably 80% or more of the saturated oxygen concentration.
The muscle tissue anchors may be coated with a biocompatible material to improve adhesion of skeletal myoblasts and prevent the muscle tissue from falling off during culture. Examples of the biocompatible material include fibronectin.
According to the culture method of the present invention, myotubes are formed when the culture of skeletal myoblasts is continued.
The hydrogel for use in culture of skeletal myoblasts may be a hydrogel of fibrin, fibronectin, laminin, collagen (e.g. types I, II, III, V, and XI), agar, agarose, a glycosaminoglycan, hyaluronic acid, a proteoglycan, and other components constituting an extracellular basement membrane matrix. The hydrogel for use may also be a commercial product. Examples include components based on a mouse EHS tumor extract (containing type IV collagen, laminin, heparan sulfate proteoglycan, etc.) sold under the trade name “Matrigel.”
As used herein, the term “collagen” encompasses undenatured collagen and denatured collagen. Examples of denatured collagen include gelatin.
The hydrogel preferably contains collagen, preferably undenatured type I collagen, in particular, when the skeletal myoblasts are derived from a bovine. The content of type I collagen, when contained, is preferably 0.3 mg/mL or more, more preferably 1.0 to 3.0 mg/mL, and still more preferably 1.0 to 1.5 mg/mL.
In terms of the skeletal myoblasts in the hydrogel, for example, the cell concentration is about 1.0×106 cells/ml or more, preferably about 1.0×107 cells/ml to about 1.0×108 cells/ml, and more preferably 5.0×107 cells/ml to about 1.0×108 cells/ml.
The skeletal myoblasts in the hydrogel can be prepared according to known methods. For example, primary myoblasts obtained by subjecting biological muscle tissue to degradative enzyme (e.g., collagenase) treatment may be used. Primary myoblasts are preferably filtered to remove impurities, such as connective tissue.
The skeletal myoblasts for use may also be cells induced to differentiate from stem cells with pluripotency, such as ES cells and iPS cells, or from somatic stem cells with an ability to differentiate into skeletal myoblasts.
Skeletal myoblasts are derived from vertebrates, such as mammals, birds, reptiles, amphibians, and fish species. Examples of mammals include non-human mammals, such as monkeys, bovines, horses, pigs, sheep, goats, dogs, cats, guinea pigs, rats, and mice. Examples of birds include ostriches, chickens, ducks, and sparrows. Examples of reptiles include snakes, crocodiles, lizards, and turtles. Examples of amphibians include frogs, newts, and salamanders. Examples of fish species include salmon, tuna, sharks, sea bream, and carp. When the three-dimensional muscle tissue is for edible use, skeletal myoblasts are preferably derived from mammals bred for animal husbandry, such as bovines, pigs, sheep, goats, and horses, and more preferably derived from a bovine.
The skeletal myoblasts for use may be skeletal myoblasts that have been genetically modified by homologous recombination, CRISPR/Cas9, or other genome editing methods, or skeletal myoblasts that have not been genetically modified. In one embodiment of the case in which the three-dimensional muscle tissue is for edible use, the skeletal myoblasts for use are preferably non-genetically modified skeletal myoblasts, from the standpoint of safety and consumer preference.
The culture medium may contain medium components (e.g., various amino acids, inorganic salts, and vitamins), serum components (e.g., growth factors, such as IGF-1, bFGF, insulin, and testosterone), antibiotics, and the like.
In the present invention, a three-dimensional muscle tissue primarily refers to an artificially produced muscle that is not derived from organisms. The three-dimensional muscle tissue of the present invention is formed of skeletal muscle cells (striated muscle cells). Skeletal muscle cells are in the foam of myotubes (myotube cells) or muscle fibers obtained through multinucleation of their precursors, i.e., myoblasts.
Typically, muscle fibers contain as a constituent unit myofibrils composed of filaments of actin, which is a protein of muscle (actin filaments), and filaments of myosin, which is a protein of muscle (myosin filaments). Myofibrils have a structure in which multiple sarcomere structures are repeated in the long-axis direction. Contraction and relaxation of muscles are known to occur based on the interaction (sliding) between actin and myosin in sarcomeres.
The three-dimensional muscle tissue of the present invention preferably has a sarcomere structure. However, no limitation is imposed on whether sliding occurs in the sarcomere structure.
Whether the three-dimensional muscle tissue has a sarcomere structure can be evaluated by known techniques. For example, the presence of sarcomeric α-actinin (SAA), which is a protein of the Z-membrane of the sarcomere structure, is evaluated by SAA immunostaining. When the SAA immunostaining is positive, and when SAA is distributed in regular stripes, the muscle tissue can be determined to have a sarcomere structure.
In the three-dimensional muscle tissue of the present invention, the muscle fibers are aligned and oriented in the same direction. The orientation of muscle fibers can be evaluated, for example, by SAA immunostaining.
When the three-dimensional muscle tissue of the present invention is for edible use, the components (preferably all components) for use in the production method of the present invention are preferably, but not limited to, components that satisfy predetermined standards and whose safety is ensured for production of food and/or for edible use.
The culture of skeletal myoblasts can be performed, for example, in the medium for proliferation culture described above by techniques known to those skilled in the art. For example, culture may be suitably performed under conditions of about 37° C. and a carbon dioxide concentration of about 5 to 10% (v/v). However, the technique is not limited to this. The culture under such conditions can be performed, for example, in a known CO 2 incubator.
The culture medium for proliferation culture may be a medium obtained by supplementing a general-purpose liquid medium, such as Dulbecco's modified Eagle's medium (DMEM), Eagle's minimal essential medium (EMEM), or alpha modified minimum essential medium (αMEM) with a serum component (e.g., horse serum, fetal bovine serum (FBS), or human serum), a component such as a growth factor, and an antibiotic such as penicillin or streptomycin.
When a serum component is added to the medium for proliferation culture, fetal bovine serum may be used as the serum component. The concentration of the serum component may be about 10% (v/v).
The culture duration may be, for example, about 1 day to 2 weeks.
The production method of the present invention can induce differentiation of skeletal myoblasts into myotubes. This differentiation induction causes multinucleation of skeletal myoblasts through cell fusion with surrounding cells to form myotubes. The myotubes further mature to form muscle fibers.
The culture above can be performed, for example, by a technique known to those skilled in the art in a medium for induction of differentiation (medium for multinucleation). For example, culture may be suitably performed under conditions of about 37° C. and a carbon dioxide concentration of about 5 to 10% (v/v). However, the technique is not limited to this. The culture under such conditions can be performed, for example, in a known CO2 incubator.
When nutrients are scarce, myoblasts are known to undergo multinucleation while engulfing surrounding cells. Thus, induction of differentiation into myotubes is preferably performed in a medium with fewer nutrients than the medium used for the proliferation culture. Horse serum, which is known to contain fewer nutrients than fetal bovine serum, may be used. The concentration of the serum component may be about 2% (v/v).
As shown in
The three-dimensional muscle tissue of the present invention does not necessarily contain heme. According to the present invention, heme is not required to supply oxygen to the cells. Heme that is slightly incorporated during cell harvesting or that is added for coloring or flavoring, rather than for oxygen supply, is regarded as an additive and is not regarded as heme in the present invention.
The present invention is described in more detail below with reference to Examples; however, the invention is, of course, not limited to the following Examples.
(I) Creation of Support Members with Anchors
As shown in
The muscle tissue anchors 11 as used here fix a shrinking muscle tissue. The support members 8 and 9 supply nutrition to the interior of the muscle tissue through a culture medium. A first connector 4 and a second connector 5 fix the muscle tissue anchor 11 and the first support members 8 or the second support members 9.
As shown in
The support members with anchors shown in
(II) Injection of Hydrogel Containing Skeletal Myoblasts (C2C12) into a Device and Culture (Parallel Culture or Vertical Culture)
As shown in
The muscle tissue anchors 11, the first support members 8, and the second support members 9 were coated with PMBV631 (2 wt % ethanol), followed by coating overnight with fibronectin (10 μL), which promotes cell adhesion. The acupuncture needles were coated by immersion in bovine serum albumin (BSA, 1%) for 1 hour to inhibit cell adhesion. For cells, C2C12 myoblasts were used. The cells (2.0×106) seeded in a 150-mm dish and passaged after 2 days were used as the cells; the cells were all P11 (passaged 11 times) or less. The cell concentration was 4.0×107 cells/ml.
In parallel culture, a hydrogel of 100% collagen gel (I-AC, AteloCell) was used, and in vertical culture, a hydrogel of a mixture of matrigel (Matrigel, Corning) and the collagen gel in a ratio of 1:1 (weight ratio), or a hydrogel of 100% matrigel was used.
The hydrogel containing skeletal myoblasts (C2C12) was injected into the device as follows.
After 3 mL of trypsin was added to a dish in which C2C12 was proliferated, incubation was performed at 37° C. for 5 minutes, and the cells were detached from the dish. After 7 mL of Dulbecco's modified Eagle's medium (below, “DMEM”) was added to the dish, pipetting was performed twice with an electric pipette to collect in 50 mL tube. Centrifugation was performed for 3 minutes (200 g) to precipitate the cells. Subsequent operations were conducted on ice.
A specific process for parallel culture is described below. Parallel culture refers to culture performed by disposing channels 3 perpendicular to the direction of gravity.
1. Two support members with anchors are positioned opposite to each other, and a first connector 4 and a second connector 5 are fixed to a substrate 17.
2. Acupuncture needles (SJ-217, produced by Seirin Corporation) are inserted into the first support members and the second support members, and side walls 18 are attached to the substrate 17 to form a culture space 2.
3. A hydrogel containing C2C12 is injected into the culture space, and incubation is performed at 37° C. for 30 minutes to solidify the hydrogel.
4. The device obtained in step 3 is placed in a 25-mL tube, and the device is immersed in a culture medium and allowed to stand for 30 minutes.
5. The acupuncture needles are removed to form channels 3.
6. The device obtained in step 5 is transferred to a dish, and the side walls 18 are removed. 7. The holes for needle insertion on the side of a culture medium inlet 6 are blocked with a sealing material 20.
8. One perfusion tube is connected to the culture medium inlet 6, and the other perfusion tube is placed near the liquid surface to perfuse the culture medium. Each of the two perfusion tubes is connected to a syringe pump, and the culture medium is perfused with the syringe pumps.
In step 8 above, the other perfusion tube is inserted below the liquid surface of the culture medium. In another embodiment, the other perfusion tube may be connected to a culture medium outlet 7.
A specific process for vertical culture is described below. Vertical culture refers to culture performed by disposing the channels 3 parallel to the direction of gravity.
1. Two support members with anchors are positioned opposite to each other, and a first connector 4 and a second connector 5 are fixed to a substrate 17.
2. Acupuncture needles (SJ-217, produced by Seirin Corporation) are inserted into the first support members and the second support members, and side walls 18 are attached to the substrate 17 to form a culture space 2.
3. A hydrogel containing C2C12 is injected into the culture space, and incubation is performed at 37° C. for 30 minutes to solidify the hydrogel.
4. One perfusion tube is connected to a culture medium inlet 6.
5. The device obtained in step 4 is placed in a 100-mL tube, and electrodes are brought close to the tube.
6. The 100-mL tube is filled with a culture medium and allowed to stand for 30 minutes.
7. The acupuncture needles are removed to form channels 3.
8. The side walls 18 are removed.
9. The holes for needle insertion on the side of the culture medium inlet 6 are blocked with a sealing material 20.
10. The other perfusion tube is placed near the liquid surface to suck up and perfuse the culture medium. Each of the two perfusion tubes is connected to a syringe pump, and the culture medium is perfused with the syringe pumps.
The vertical culture was performed by rotating the tube at 60 rpm by shaking the tube with a shaker. By creating convection flows in the culture medium within the tube, the concentration of oxygen, which can only be taken in from the liquid surface of the culture medium, can be kept constant within the culture medium. As shown in
To promote maturation of myotubes, electrical stimulation of 0.5 V/mm was applied to the cultured product for 2 hours while maintaining the temperature and carbon dioxide concentration in the device constant from day 4 after the start of culture.
After completion of culture, tissue sections of the three-dimensional muscle tissue were created.
The sections were prepared in the following order: (i) cryoprotection treatment, (ii) freeze treatment, (iii) creation of sections, (iv) HE staining, and (v) immunostaining.
Cryoprotection treatment is a treatment for preventing ice grains from being formed in the tissue when frozen. In the following description, PBS refers to phosphate buffered saline.
1. After the cultured product (three-dimensional muscle tissue) was washed once with PBS(−), the culture product was immersed for one day each in cryo-dishes containing a 10% sucrose/PBS(−) solution, 20% sucrose/PBS(−) solution, or 30% sucrose/PBS(−) solution.
2. For the washing treatment and immersion treatment in 10%, 20%, and 30% sucrose/PBS(−) solutions, the three-dimensional muscle tissue detached from the anchors was used.
3. The muscle tissue was immersed in a cryo-dish filled with an OCT compound agent and allowed to stand at room temperature for 2 days.
Freeze treatment was performed before the creation of frozen sections.
1. Liquid nitrogen was poured into an insulated container (height: 4 to 5 cm).
2. The cryo-dish was placed into the liquid nitrogen by using tweezers.
3. Since long-time immersion in liquid nitrogen causes cracking in the three-dimensional muscle tissue, the cryo-dish was moved in and out of the liquid nitrogen every 1 to 2 seconds to gradually solidify the three-dimensional muscle tissue.
(iii) Creation of Sections
Sections with a thickness of 8 μm were created using a cryostat. The details are described as follows.
1. The cryo-dish containing the three-dimensional muscle tissue sample after freeze treatment was placed in a cryostat and warmed to −20° C.
2. The sample was collected from the cryo-dish, and an 8-μm-thick short-side section was created.
3. The sample was rotated 180° to create an 8-μm-thick short-side section on the opposite side.
4. The sample was rotated 90° to create three to four 8-μm-thick long-side sections.
The sample was shaved (200 μm), and long-side sections were repeatedly created until no more long-side sections were created.
6. The resulting sections were attached to MAS-coated glass slides.
(iv) HE staining
HF staining was performed as follows.
1. After the section was prepared, the section was dried for 1 day.
2. The section was immersed in a Mayer's hematoxylin solution and allowed to stand for 5 minutes.
3. Excess hematoxylin solution was removed through adsorption on paper.
4. The section was transferred to a staining bottle containing water to remove excess staining solution.
5. The section was immersed in warm water at 50° C. and allowed to stand for 5 minutes.
6. The section was transferred to a staining bottle containing water.
7. The section was immersed in an eosin solution and allowed to stand for 5 minutes.
8. The slide glass was immersed in 100% ethanol and slowly moved up and down 5 times to remove excess staining solution.
9. The operation in step 8 was performed three times in total using different bottles.
10. The resulting product was immersed in xylene (3 times for 5 minutes each).
11. A few drops of Entellan were added to the section for sealing, and a cover glass was attached.
12. The resulting product was dried overnight.
Immunostaining was performed as follows.
1. A circle was drawn around the section with a PAP pen.
2. An FBS solution (1% PBS) was added dropwise to the inside of the circle drawn with the PAP pen and allowed to stand for 1 hour for blocking.
3. The resulting product was placed in a staining bottle such that the section was not peeled off, followed by washing with PBS.
4. A primary staining solution was added dropwise to the inside of the circle drawn with the PAP pen and allowed to stand for 1 hour.
5. The resulting product was placed in a staining bottle such that the section was not peeled off, followed by washing with PBS.
6. A secondary staining solution was added dropwise to the inside of the circle drawn with the PAP pen and allowed to stand for 1 hour.
7. The resulting product was placed in a staining bottle such that the section was not peeled off, followed by washing with PBS.
8. DAPI was added dropwise.
9. A cover glass was placed on.
10. Observation was conducted from the cover glass side.
The three-dimensional muscle tissue prepared according to the present invention is characterized by being oriented.
The orientation index of the three-dimensional muscle tissue was evaluated as follows using two-dimensional Fourier transform.
1. The image was cut so that the length and width are power of 2 pixels.
2. The image was grayed.
3. The Hanning window was used.
4. Two-dimensional Fourier transform was applied using the fft2 function of a numerical analysis software (MATLAB (registered trademark), provided by The MathWorks, Inc.).
5. The 2D Fourier transform image was integrated in polar coordinates, and the pixel values from 1 degree to 180 degrees were added.
6. Each value from 1 degree to 180 degrees was divided by the sum of these values to obtain the average.
Furthermore, nuclei were identified in the HE-stained images using image processing software (ImageJ, open source), and the number of nuclei were counted as follows.
1. The images were combined using Make composite.
2. The image were converted to 32-bit RGB color using RGB Color values.
3. The RGB color image was split for HE using Deconvolution.
4. The threshold was applied to the blue image to separate the image containing the nuclei from the background.
5. The connected nuclei was separated using Watershed.
6. The number of nuclei was counted.
The shape of the channels 3 and the flow in the channels 3 were confirmed.
A gel structure without cells was produced using a collagen gel as the hydrogel and using the nine-support-member array shown in
To confirm that the channels were not blocked, ink was allowed to flow through the channels.
Specifically, with support members, a gel structure without cells was produced using a collagen gel, and blue ink was allowed to flow immediately after gelation. The appearance of flow was recorded on video through a microscope (STZ-171, Shimadzu Corporation).
As shown in
This clarified that delivering a culture medium that contains oxygen and nutrients into the interior of the three-dimensional muscle tissue is possible.
First, the difference between vertical culture and parallel culture is explained. In the case of parallel culture, when 100% collagen gel was used, an alginate gel poured from the culture medium inlet 6 after 7 days of culture was discharged from the culture medium outlet 7. However, when a gel mixture of collagen gel and matrigel (collagen gel:matrigel=50%:50%) was used, an alginate gel poured from the culture medium inlet 6 after 7 days of culture was not discharged from the culture medium outlet 7. This is presumably because the channels were blocked during the 7 days of culture since matrigels are softer than collagen gels and thus easily cause deformation of the structure by gravity.
In the case of vertical culture, the direction of gravity is the same as that of the channels, making it less likely to cause blockage of the channels; thus vertical culture is more suitable than parallel culture. The details are described later.
A gel structure was produced in the same manner as described above using a four-support-member array and using a cell-free hydrogel obtained by mixing a collagen gel and matrigel in a ratio of 1:1. The resulting gel structure was placed in DMEM medium and cultured in an incubator for 1 day.
As shown in
These results suggest that the ink flow observed at the bottom of the images of parallel culture was due to the ink that could not flow into the channels from the culture medium inlet 6 and thus leaked out from the gap between the support members and the gel. Focusing on the support member on the lower side of 0.09 S and 0.18 S images in (b), it is confirmed that bubbles accumulated inside the support member were about to flow into the channel but unable to do so. This is presumably because the channel was blocked.
The results of parallel culture using 100% collagen gel are described here. The purpose of this experiment is to show that culture of muscle tissue is possible even by parallel culture if a collagen gel is used alone. In this experiment, when an alginate gel was allowed to flow into the channels after 7 days of culture, red culture medium, which was assumed to have remained in the channels, was pushed out from the opposite side of the channels, indicating that the channels remained open.
The degree of spontaneous shrinkage of tissue during culture was confirmed to vary depending on whether the tissue was with or without perfusion. Specifically, after 36 hours of culture, the tissue with perfusion showed 39% shrinkage in width compared to the original (
To confirm the orientation index of the muscle tissue, the periphery of the muscle tissue was stained with a fluorescent dye for observation.
After 36 hours of culture, the muscle tissue was cut and subjected to live/dead assay.
Another characteristic result was that more viable cells were present between the channels while more dead cells were present around the channels. The lack of oxygen in culture is a possible cause of cell death. In this case, cells closer to the channels should be viable, and dead cells should increase as the distance from the channels increase; however, the trend here was the opposite. This suggests that the cells did not die during the culture period, but that the cells around the channels were damaged and died when the acupuncture needles were pulled out to foam the channels. Since the needles were coated with BSA, cell adhesion to the needles was unlikely to occur. However, the cell death was possibly caused due to rubbing when the needles were removed. To avoid this, the needles must be removed slowly.
To confirm whether the support members 8, 9 and the anchors 11 function in vertical culture, the appearance of the cells after culture was observed using HE staining and fluorescent immunostaining images.
Muscle tissue has properties of shrinking during culture and thus must be anchored.
(i) The percentage of shrinkage after 7 days of culture increased in the following order: w/perfusion, w/o perfusion, and w/o anchor.
(ii) In some samples of w/o perfusion, the muscle tissue fell off from the support members.
The results in (i) above can be explained according to the number of viable cells. It is well known that muscle tissue shrinks during the culture period. The shrinkage occurs due to the traction of the cells reorganizing the extracellular matrix.
Due to the effect of perfusion, it is believed that more cells were alive in the tissue w/perfusion than in the tissue w/o perfusion; thus, the amount of cells involved in shrinkage was also believed to be greater in the tissue w/perfusion. It is thus believed that the degree of shrinkage in width was greater in the tissue w/perfusion than the tissue w/o perfusion.
Next, the order, i.e., w/o perfusion and then w/o anchor, was believed to be due to the difference between presence and absence of displacement in the vertical direction. Between the tissue w/o perfusion and the tissue w/o anchor, the presence or absence of the anchors made a difference in the degree of shrinkage in width. The tissue with anchors (w/o perfusion) showed no displacement in the vertical direction, while the tissue w/o anchor showed displacement at the edge of the tissue even to the tip of the support members. Without the effect of perfusion, the percentages of shrinkage would have been the same between these two; however, presumably, due to the presence of displacement in the vertical direction, the tissue w/o anchor resulted in a smaller degree of shrinkage in width.
Regarding (ii) above, as shown in
These results indicate that the anchors 11 prevent the muscle tissue from falling off in the device of the present invention for culturing a contractile muscle tissue.
Channels after Culture
Whether the channels 3 remained open after culture even when shrinkage occurred, and whether the culture medium was perfused, were confirmed by allowing ink to flow. After 7 days of culture, ink was allowed to flow from the bottom to the top of the channels 3 using the tube through which a culture medium had been flowing (
The presence of channels was also confirmed in a long-side section prepared using a cryostat from a sample different from that mentioned above (
An 8-μm-thick section was prepared parallel to the cross-section perpendicular to the channels, and HE staining was performed. The reason that the nuclei were not present in the center is explained here.
From the binarized image in
On the other hand, the initial cell concentration of the sample used in this experiment was 4.0×107 cells/ml. Considering that the cross-section (16 mm2) shrank to about 1.3 mm2, the cell concentration would be 49×107 cells/ml.
Accordingly, the cell concentration based on actual measurement and the cell concentration estimated from the initial cell concentration of the sample are almost the same, suggesting that the nuclei at the center have moved.
Next,
Relationship Between the Distance from the Interface Between the Tissue and Culture Medium and the Cell Concentration
The cell concentrations were compared between the muscle tissue sample with perfusion and the muscle tissue sample without perfusion (
Dense Nuclei when Perfusion Succeeded
In the sample in which perfusion succeeded, the nuclei were densely packed around the channels (
The orientation index of actin filaments of the tissue cultured for 8 days without shaking was studied. The images in the upper row of
Number | Date | Country | Kind |
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2020-154316 | Sep 2020 | JP | national |
Filing Document | Filing Date | Country | Kind |
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PCT/JP2021/032244 | 9/2/2021 | WO |