JP6474219B2 - Use of cell culture substrate, cell culture substrate with cell, method for producing cell culture substrate with cell, cell culture method, use of cell culture device, and cell culture device - Google Patents

Description

  The present invention relates to a cell culture substrate, a method for producing a cell culture substrate, a cell culture method, and a cell culture apparatus.

Currently, the most common cause of death in Japanese is malignant neoplasm (cancer). Three major therapies, such as surgical therapy, chemotherapy, and radiation therapy, are mainly used to treat cancer, but these therapies have been reported to have adverse effects and adverse effects on the human body. There is concern about a decline in quality of life (QOL). Therefore, "immunotherapy" is currently attracting attention as a cancer treatment method that is expected to improve QOL. This therapy is expected as a novel cancer treatment method because it has few side effects on the human body and does not select the site of cancer.
The devised immunotherapy is roughly classified into two types. One is called “active immunotherapy” and is a method of treating cancer by activating immune cells by incorporating cytokines or immunostimulants into the patient's body.
The other type of immunotherapy is called “adoptive immunotherapy”, in which the patient's own immune cells are cultured, propagated or activated outside the body, and the immune cells in the body are activated by returning them to the patient. How to treat cancer. Adoptive immunotherapy has the advantage of being able to cure the disease by enhancing the patient's resilience without using expensive cytokines. Therefore, the appearance of a cell culture substrate capable of culturing immune cells outside the body and having an immunostimulatory effect is demanded.

Inositol phosphate is obtained by phosphorylating the hydroxyl group of inositol. Examples of inositol phosphates include inositol monophosphate (IP), inositol diphosphate (IP ₂ ), inositol triphosphate (IP ₃ ), and inositol pentaphosphate (IP ₅ ) depending on the number of phosphate esterified hydroxyl groups. ), Inositol hexaphosphate (IP ₆ ) (also called phytic acid) is known. Inositol hexaphosphate (IP ₆ ) has been reported to have an anticancer effect and a function of activating immune cells (Non-patent Document 1).

A.M.Shamsuddin, I. Vucenik, K. E. Cole, Life Science, 61, 343-354 (1997).

  The present invention has been made in view of the above circumstances, a cell culture substrate capable of culturing cells such as immune cells, a method for producing the cell culture substrate, a cell culture method using the cell culture substrate, and An object is to provide a cell culture device.

  The present invention provides a cell culture substrate having the following characteristics, a method for producing the cell culture substrate, a cell culture method using the cell culture substrate, and a cell culture apparatus.

(1) the small without even surface, including the metal compound that inositol phosphate or its salt are combined,
The cell culture substrate , wherein the metal compound is a ceramic of a calcium phosphate compound ,
Use to culture T cells in contact with outside the body .
(2) The use according to (1), wherein the inositol phosphate is inositol hexaphosphate.
(3) At least the surface has a metal compound to which inositol phosphate or a salt thereof is bound,
A cell culture substrate in which the metal compound is a ceramic of a calcium phosphate compound;
T cells in contact with the cell culture substrate;
A cell culture substrate with a cell.
(4) The cell culture substrate with a cell according to (3), wherein the inositol phosphate is inositol hexaphosphate.
( 5 ) The cell-attached cell according to (3) or (4), wherein a solution containing inositol phosphate or a salt thereof is brought into contact with a metal compound, and the T cell is brought into contact with the obtained cell culture substrate. A method for producing a culture substrate.
( 6 ) At least the surface has a metal compound to which inositol phosphate or a salt thereof is bound, and the T compound is brought into contact with a cell culture substrate in which the metal compound is a ceramic of a calcium phosphate compound. A cell culture method for culturing.
(7) At least the surface has a metal compound to which inositol phosphate or a salt thereof is bound,
A cell culture apparatus comprising a cell culture substrate in which the metal compound is a ceramic of a calcium phosphate compound, and a culture tank for containing the cell culture substrate, for bringing T cells into contact with each other and culturing them outside the body. use.
(8) The use according to (7), wherein the inositol phosphate is inositol hexaphosphate.
(9) pre-SL (3) or (4) a cell with the cell culture substrate according to the previous SL cell culture device comprising a culture tank for containing the cells with cell culture substrate.

  According to the present invention, a cell culture substrate capable of culturing cells such as immune cells, a method for producing the cell culture substrate, a cell culture method using the cell culture substrate, and the cell culture substrate are provided. A cell culture device can be provided.

It is a mimetic diagram showing one embodiment of a cell culture substrate concerning the present invention. It is a mimetic diagram showing one embodiment of a cell culture substrate concerning the present invention. It is a schematic diagram which shows an example of the mode of cell culture of the cell culture method of this invention and another method. It is a block diagram which shows one Embodiment of the cell culture apparatus of this invention. In an Example, it is a graph which shows the result of having measured the surface roughness and relative density of the hydroxyapatite (HAp) ceramics. In embodiments, a graph showing the results of analysis of X-ray diffraction of HAp Ceramics surface modifying IP _{6 (IP 6} -HAp ceramics) (XRD). In _embodiments, a graph showing the results of analysis of IP 6 -HAp X-ray photoelectron spectroscopy of the ceramic (XPS). In the examples, analyzes of IP ₆ -HAp X-ray photoelectron spectroscopy of the ceramic (XPS), is a graph showing a result of comparing the narrow spectrum. In the examples, analyzes of IP ₆ -HAp X-ray photoelectron spectroscopy of the ceramic (XPS), compares the narrow spectrum is a graph showing a result of further performing peak separation. It is a graph which shows the result of the increase / decrease rate of a helper T cell when cell culture is performed in an Example. It is a graph which shows the result of the increase / decrease rate of a killer T cell at the time of performing cell culture in an Example.

≪Cell culture substrate≫
The cell culture substrate of this embodiment has a metal compound to which inositol phosphate or a salt thereof is bound at least on the surface.

Inositol phosphate is a phosphate esterified from at least one of the six hydroxyl groups of inositol (1,2,3,4,5,6-cyclohexanehexaol). In the present embodiment, inositol phosphoric acid in which three or more of the hydroxyl groups are phosphate ester is preferable, inositol phosphate in which four or more of the hydroxyl groups are phosphate ester is more preferable, and all the hydroxyl groups of inositol are phosphate esters. Most preferred is a modified phytic acid (inositol hexaphosphate, IP ₆ ). Inositol phosphates may be used alone or in combination of two or more.

Moreover, as long as it has an immunostimulatory effect, even if it is a salt of inositol phosphate, it is good also as a component of the cell culture base material of this embodiment similarly to inositol phosphate.
The salt of inositol phosphate is preferably an alkali metal salt or an alkaline earth metal salt, such as a sodium salt, potassium salt, magnesium salt, calcium salt, barium salt, and more preferably an alkali salt such as sodium salt or potassium salt. It is a metal salt. Among these, inositol hexaphosphate sodium salt and inositol hexaphosphate potassium salt are particularly preferable. As inositol hexaphosphate sodium salt, hydrates having different crystallization water contents are known, and any of them can be preferably used. Moreover, the salt of inositol phosphate may be used alone or in combination of two or more.

  The metal compound is a compound containing a metal element, and may be an organic metal compound or an inorganic metal compound, preferably an inorganic metal compound. Inositol phosphate has a strong chelating action. Inositol hexaphosphate is known to form chelate bonds with various metal ions. Metal ions known to form chelate bonds with inositol hexaphosphate include magnesium ions, calcium ions, potassium ions, silver ions, iron ions, zinc ions, and the like. Therefore, the metal compound and inositol phosphate can be bound by a chelate bond.

The metal element contained in the metal compound is preferably a Group 2 element. The Group 2 element is an element classified into Group 2 of the periodic table, and examples thereof include magnesium, calcium, strontium, and barium, and calcium is preferable.
The metal compound is preferably a ceramic. Ceramics is a stable material and is easy to sterilize. The ability to reduce the risk of contamination of microorganisms and the like by sterilization is very useful as a cell culture substrate.
Therefore, the metal compound is more preferably a ceramic containing a Group 2 element.

The metal compound is preferably a calcium phosphate compound. Typical calcium phosphate compounds include hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ; HAp), α-tricalcium phosphate, β-tricalcium phosphate, tetracalcium phosphate, octacalcium phosphate , Calcium hydrogen phosphate, calcium dihydrogen phosphate, amorphous calcium phosphate, and fluoroapatite, preferably hydroxyapatite, α-tricalcium phosphate, β-tricalcium phosphate, more preferably hydroxyapatite It is. Apatite is an inorganic crystalline substance of phosphate, and is excellent in biocompatibility.

  The metal compound included in the cell culture substrate of the present embodiment is more preferably a calcium phosphate compound ceramic containing calcium as a constituent element. By using the metal compound as a ceramic of a calcium phosphate compound containing calcium as a constituent element, a cell culture substrate excellent in sterilization ease, immune cell culture performance, and inositol phosphate binding performance can be obtained. Furthermore, since it has high biocompatibility, it is more preferable that it is a ceramic of hydroxyapatite.

The shape of the cell culture substrate is not particularly limited, and may be any shape such as granular, fine particle, sheet, plate, disk, rod, column, and polygonal column. As long as it has a metal compound to which inositol phosphate or a salt thereof is bound at least on the surface, for example, the entire cell culture substrate is composed of the metal compound, and inositol phosphate or a salt thereof is bound to the surface. The substrate surface layer may be composed of the metal compound, and the interior of the substrate may be composed of something other than the metal compound.
The cell culture substrate may be dense or porous. In the case of being porous, inositol phosphate may be disposed inside the pore as long as it has a metal compound to which inositol phosphate or a salt thereof is bound at least on the surface.

FIG. 1 is a schematic view showing one embodiment of a cell culture substrate according to the present invention. The cell culture substrate 1 has a structure in which inositol phosphate 4 is bonded to a disk-shaped metal compound 2.
FIG. 2 is a schematic view showing one embodiment of the cell culture substrate according to the present invention. The cell culture substrate 11 has a hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ) layer 12 formed on the surface of the central layer 13, and inositol phosphate 4 is bound to the hydroxyapatite layer 12. It has a structure.

  As shown in FIG. 1 and FIG. 2, the cell culture substrate according to the present invention has at least a metal compound having inositol phosphate bonded to the surface thereof, so that the cell culture substrate can be used for cell culture. When cells are brought into contact with cells, the cells come into contact with inositol phosphate, and the cells are efficiently activated and immunity can be activated.

≪Method for producing cell culture substrate≫
In the method for producing a cell culture substrate of the present invention, a metal compound is brought into contact with a solution containing inositol phosphate or a salt thereof.

  A solution containing inositol phosphate is obtained by mixing a liquid such as water or various buffer solutions with inositol phosphate. The pH of the solution containing inositol phosphate may be about pH 6-8.

  The concentration of inositol phosphate in the solution containing inositol phosphate is preferably 500 ppm (w / v) or more, and more preferably 2500 ppm (w / v) or more. The upper limit of the concentration of inositol phosphate in the solution is not particularly limited, and may be a saturated concentration. However, in the examples described later, when the metal compound is immersed in a solution containing 3000 ppm (w / v) or more of inositol phosphate, the binding amount of inositol phosphate to the metal compound is considered to be saturated. Since a tendency was observed, the concentration of inositol phosphate in the solution containing inositol phosphate is preferably 500 ppm (w / v) or more and 6000 ppm (w / v) or less, and 2500 ppm (w / v). More preferably, it is 6000 ppm or less, and further preferably 2500 ppm (w / v) or more and 4000 ppm (w / v) or less.

  The method for bringing the metal compound into contact with the solution containing inositol phosphate is not particularly limited, and the metal compound is coated with a solution containing inositol phosphate, and the metal compound is sprayed with a solution containing inositol phosphate. Examples thereof include a method of immersing a metal compound in a solution containing inositol phosphate.

Among the methods listed above, it is preferable to immerse the metal compound in a solution containing inositol phosphate. In this case, the time for immersing the metal compound in the solution containing inositol phosphate is preferably 0.1 hour to 100 hours, more preferably 1 hour to 75 hours, and still more preferably 10 hours to 50 hours. The temperature of the solution containing inositol phosphate is preferably 10 ° C to 60 ° C, more preferably 20 ° C to 50 ° C, and particularly preferably 30 ° C to 40 ° C.
The method of immersing the metal compound in the solution containing inositol phosphate is not particularly limited. For example, the method of adding inositol phosphate in water after immersing the metal compound in water, adding inositol phosphate to water A method of immersing a metal compound in the water later can be exemplified.
The state of the immersion is not particularly limited. For example, the metal compound is allowed to stand in the solution, the metal compound is stirred in the solution, and the solution is added to the metal compound that is left in the solution. It can be performed by a method such as stirring.

  When a solution containing inositol phosphate or a salt thereof is brought into contact with the metal compound, the inositol phosphate forms a chelate bond with the metal element contained in the metal compound, and the inositol phosphate is bonded to the surface of the metal compound.

In the method for producing a cell culture substrate of the present invention, inositol phosphate or a salt thereof that is not bound to a metal compound is obtained from a substrate obtained by bringing a metal compound into contact with a solution containing inositol phosphate or a salt thereof. You may have the washing process for removing. In the production method, the method having the washing step is preferable from the viewpoint of preventing inhibition of cell activation by a high concentration of inositol phosphate that is not bound to a metal compound.
As the washing step, for example, a substrate obtained by bringing a metal compound into contact with a solution containing inositol phosphate or a salt thereof, than a solution not containing inositol phosphate, or the above solution in contact with a metal compound Examples of the liquid not containing inositol phosphate include water, various buffer solutions, and the like.

  The description relating to the inositol phosphate is the same for the salt of inositol phosphate.

≪Cell culture method≫
The cell culture method of the present invention comprises culturing the cells by bringing the cells into contact with the cell culture substrate of the present invention. By bringing the cells into contact with the cell culture substrate of the present invention, the cells come into contact with the inositol phosphate or salt thereof bound to the metal compound of the cell culture substrate.

  FIG. 3 is a diagram schematically showing an example of a state of cell culture in the cell culture method of the present invention and another method. FIG. 3A shows a case where cell culture is performed in a liquid medium containing inositol phosphate. FIG. 3B is an example of the cell culture method of the present invention, and shows a case where cell culture is performed on a cell culture substrate having a metal compound to which inositol phosphate is bound.

As shown in Reference Examples 3 to 12 described later, the inventors used this culture system as shown in FIG. 3A to cultivate cells in a liquid medium containing inositol phosphate. It has been found that good cell culture cannot be achieved unless the concentration of inositol phosphate present in the cell culture system (cell culture medium) is high compared to the cell culture method of the invention. In addition, for the first time, it has been clarified in this application that the concentration of inositol phosphate present in the cell culture system (cell culture solution) is sometimes too high, which may hinder good cell culture.
In contrast, in the cell culture method of the present invention shown as an example in FIG. 3 (b), the inventors cultivate cells on a substrate to which inositol phosphate is bound, whereby FIG. It was found that better cell culture was achieved with a small amount of inositol phosphate bound to the substrate as compared to the culture system as shown in FIG. In addition, even if a small amount of inositol phosphate is used, good cell culture is achieved, and there is no possibility that cell culture will be hindered. The amount of inositol phosphate bound to the surface of the cell culture substrate in the culture system as shown in FIG. 3 (b) was calculated using the saturated adsorption amount of IP _{6 to} the HAp powder of 0.427 mg · m ^−2. Then, when ceramics having a diameter of 15 mm and a thickness of 1 mm is used, it becomes about 95 ng.

Consider the relationship between cells and inositol phosphates in the above cell culture.
In the culture system as shown in FIG. 3 (a), inositol phosphate is dissolved in the culture solution. At this time, the cells are considered to come in contact with inositol phosphate dissolved in the culture solution one after another. While cells are activated by reaction with inositol phosphate, it is thought that cell activation is inhibited by reaction with high concentration of inositol phosphate. And if this is the case where the concentration of inositol phosphate present in the cell culture medium is not high, good cell culture is not achieved, and the concentration of inositol phosphate is too high, which leads to good cell culture. It is thought that it will be disturbed.
On the other hand, in the culture system as shown in FIG. 3 (b), a substrate on which inositol phosphate is bound to the surface is used. At this time, since the cells come into contact with the inositol phosphate on the surface of the substrate, the cells can be perceived as having an appropriate amount of inositol phosphate in the culture system, and further, the cells by reaction with inositol phosphate. It is thought that good results are also obtained for activation of.

  The cell culture method of the present invention includes culturing cells to be cultured and the cell culture substrate of the present invention in a liquid medium. By causing the cells to be cultured and the cell culture substrate of the present invention to coexist in the same culture solution, the cells come into contact with the cell culture substrate.

The target cell cultured by the cell culture method of the present invention is not particularly limited, but the cell is preferably an immune cell, more preferably a T cell.
As immune cells, leukocytes, lymphocytes, T cells, helper T cells, killer T cells, B cells, natural killer cells (NK cells), natural killer T cells (NKT cells), granulocytes, neutrophils, eosinic acid Examples include spheres, basophils, macrophages, dendritic cells, and progenitor cells or stem cells that can differentiate into them.

  The description relating to the inositol phosphate is the same for the salt of inositol phosphate.

≪Cell culture device≫
The cell culture device of the present invention includes the cell culture substrate of the present invention and a culture tank for housing the cell culture substrate.
FIG. 4 is a configuration diagram showing an embodiment of the cell culture device of the present invention. The cell culture device 20 includes a culture tank 25, and the cell culture substrate 1 is disposed inside the culture tank. In the cell culture substrate 1, inositol phosphate 4 is bound to a disk-shaped metal compound 2.

  Since the cell culture device 20 of the present embodiment has the cell culture substrate 1 having the metal compound to which inositol phosphate is bound on at least the surface described above, the cells are added to the culture tank 25, and the culture substrate and the cells are added. Is brought into contact with inositol phosphate, the cell is efficiently activated, and immunity is activated.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated more concretely, this invention is not limited to these.

1-1. Production of HAp ceramics HAp compacts were produced according to the previous report (Z. Zhuang, TJ Fujimi, M. Nakamura, T. Konishi, H. Yoshimura and M. Aizawa, Acta Biomaterialia, 9, 6732-6740 (2013)). 1 g of HAp-100 (manufactured by Taihei Chemical) was weighed and pressure-molded under the conditions of a diameter of 21 mmΦ, a height of ˜2 mm, and a molding pressure of 50 MPa to obtain a HAp molded body. The HAp ceramic was obtained by firing this HAp compact at a temperature of 1200 ° C., a holding time of 5 hours, and a heating rate of 10 ° C./min ⁻¹ . The obtained HAp ceramic was disk-shaped having a diameter of 15 to 16 mm and a height of 1 to 2 mm.
HAp ceramics are ultrasonically cleaned in ethanol, then mirror polished with water-resistant abrasive paper (in the order of # 400, # 1000, # 2000), and the polished sample is ultrasonically cleaned again in ethanol at 160 ° C. Sterilized by dry heat for 1.5 hours.

1-1-2. Characterization of HAp ceramics The surface roughness and relative density of HAp ceramics that had undergone the above ultrasonic cleaning, mirror polishing, ultrasonic cleaning, and dry heat sterilization processes were measured. The surface roughness was Ra = about 0.1 μm, and was performed using a measuring machine (SURFTEST SV-3100 JISB0601: 2001, manufactured by Mitutoyo Corporation). The relative density was calculated by measuring the bulk density of the manufactured HAp ceramics and setting the theoretical value 3.16 g / cm ³ of the bulk density of the HAp ceramics as 100%.
The results are shown in FIG. By mirror polishing, a uniform surface with few errors between HAp ceramic samples could be obtained. It was also found that the HAp ceramic was a dense body having a relative density of 97.71%.

1-2. Preparation of IP ₆ solution 50% by weight of IP ₆ solution, IP ₆ concentrations of 1000 ppm (w / v), 2000 ppm (w / v), 3000 ppm (w / v), 5000 ppm (w / v), respectively The pH was adjusted to 7.3 with an aqueous NaOH solution to obtain an IP ₆ solution having each concentration described above. The IP ₆ solution used for the cell culture experiment was sterilized with a 0.2 μm sterilization filter.

1-3. Preparation of IP ₆ -HAp ceramics (Example 1)
Using HAwell ceramics obtained in 1-1 above, 12-well plate, 1-2. It was immersed in 3 cm ³ of the 1000 ppm (w / v) IP ₆ solution obtained in the section.
Immersion was performed for 24 hours in an incubator at 37 ° C. and 5% CO ₂ atmosphere. After immersion, the plate was washed three times with sterilized ultrapure water, dried and used as a culture substrate. The produced IP ₆ -HAp ceramics is expressed as “IP ₆ -HAp (1000)” depending on the concentration of the IP ₆ solution in which the IP ₆ -HAp ceramic is immersed.

(Example 2)
Instead of the 1000 ppm (w / v) IP ₆ solution, the HAp ceramic obtained in 1-1 above is replaced with 1-2. An IP ₆ -HAp ceramic was produced in the same manner as in Example 1 except that it was immersed in 3 cm ³ of the 2000 ppm (w / v) IP ₆ solution obtained in the section. The produced IP ₆ -HAp ceramics is expressed as “IP ₆ -HAp (2000)” according to the concentration of the IP ₆ solution in which the IP ₆ -HAp ceramic is immersed.
(Example 3)
Instead of the 1000 ppm (w / v) IP ₆ solution, the HAp ceramic obtained in 1-1 above is replaced with 1-2. An IP ₆ -HAp ceramic was prepared in the same manner as in Example 1 except that the sample was immersed in 3 cm ³ of the 3000 ppm (w / v) IP ₆ solution obtained in the section. The produced IP ₆ -HAp ceramics is expressed as “IP ₆ -HAp (3000)” according to the concentration of the IP ₆ solution immersed therein.
Example 4
Instead of the 1000 ppm (w / v) IP ₆ solution, the HAp ceramic obtained in 1-1 above is replaced with 1-2. An IP ₆ -HAp ceramic was produced in the same manner as in Example 1 except that it was immersed in 3 cm ³ of the 5000 ppm (w / v) IP ₆ solution obtained in the section. The produced IP ₆ -HAp ceramics is expressed as “IP ₆ -HAp (5000)” depending on the concentration of the IP ₆ solution in which the IP ₆ -HAp ceramic is immersed.

(Reference Example 1)
The HAp ceramic obtained in 1-1 above is immersed in 3 cm ³ of ultrapure water sterilized as an IP ₆ solution of 0 ppm (w / v) instead of an IP ₆ solution of 1000 ppm (w / v). Except for the above, IP ₆ -HAp ceramics were produced in the same manner as in Example 1 above. The produced IP ₆ -HAp ceramics is denoted as “IP ₆ -HAp (0)”.

2. To confirm the crystal structure characterization _IP 6 -HAp ceramics IP ₆ -HAp ceramics, it was analyzed using X-ray diffraction (XRD). FIG. 6 shows a spectrum obtained by XRD. In all the ceramics of Examples 1 to 4, it was confirmed to be a HAp single phase.

In order to confirm the presence of IP _{6 on} the surface of the IP ₆ -HAp ceramic, chemical state analysis was performed using X-ray photoelectron spectroscopy (XPS). FIG. 7 shows a wide spectrum in XPS. In all samples, peaks of Ca, P and O due to HAp were confirmed.
Further, FIG. 8 shows a comparison of the narrow spectrum of each sample by paying attention to the peak of carbon. In each spectrum, the value of hydroxycarbon contaminated on the surface confirmed by C 1s (285.2 eV) was used as a reference value, and the charge-up of the sample was corrected by that value.
The narrow spectrum was subjected to peak separation using a Gauss-Lorentz composite function. FIG. 9 shows a narrow spectrum of IP ₆ -HAp (5000) before and after peak separation. A small peak was observed that overlapped to the left of the large peak of C 1s that appears to be derived from impurities and the C—C and C—H bonds present in the molecular structure of IP ₆ . This peak is considered to be derived from C-O bond, it can be said that this is derived from C-O bonds present in the molecular structure of IP _6. Therefore, it is presumed that IP ₆ is present on the HAp ceramics in all the IP ₆ -HAp ceramics of Examples 1 to 4.

When the amount of IP ₆ present on the HAp ceramic was calculated using the saturated adsorption amount of IP ₆ with respect to the HAp powder of 0.427 mg · m ⁻² , it was about 95 ng.

3. Cultivation of mouse-derived spleen cells in IP ₆ -HAp ceramics (Example 5)
1.0 × 10 ⁶ cells · cm ⁻³ spleen cells derived from C57BL / 6N mice were treated as described in 1-3. The seeds were seeded on the IP ₆ -HAp (1000) obtained in the above section, and one day later, the ratio of spleen cells was examined using a flow cytometer. A 24 well polystyrene plate was used as a control. The spleen cells were subjected to Live / Dead cell staining (manufactured by life technologies), and the separated live cells were reacted with the following antibodies to separate the cells.
Anti-CD3 antibody (manufactured by BD) as a surface marker for T cells, anti-CD4 antibody (manufactured by BECKMAN) as a helper subset marker for mature T cells, anti-CD19 antibody (manufactured by BD) as a surface marker for B cells, and mature T cells An anti-CD8 antibody (manufactured by Invitrogen) was used as a cytotoxic subset marker.

(Example 6)
Culture was performed in the same manner as in Example 5 except that instead of IP ₆ -HAp (1000), spleen cells derived from C57BL / 6N mice were seeded on the above IP ₆ -HAp (2000).
(Example 7)
Culture was performed in the same manner as in Example 5 except that instead of IP ₆ -HAp (1000), spleen cells derived from C57BL / 6N mice were seeded on the above IP ₆ -HAp (3000).
(Example 8)
Culture was performed in the same manner as in Example 5 except that instead of IP ₆ -HAp (1000), spleen cells derived from C57BL / 6N mice were seeded on the above IP ₆ -HAp (5000).
(Reference Example 2)
Culture was performed in the same manner as in Example 5 except that instead of IP ₆ -HAp (1000), spleen cells derived from C57BL / 6N mice were seeded on the above IP ₆ -HAp (0).

  The results are shown in Table 1 as the rate of change relative to the percentage of cells in the control.

  The increase / decrease rate of the cells in Table 1 was calculated by the following formula using the control cells as a reference.

From the results shown in Table 1, it can be seen that the increase rates of helper T cells and killer T cells are remarkably high in IP ₆ -HAp (3000) and IP ₆ -HAp (5000).

B cells showed the opposite tendency to T cells. This indicates that the proportion of B cells in the sample has decreased relative to the control, and is also considered to be the result of an increase in the proportion of T cells.
The ratio of helper T cells and killer T cells in IP ₆ -HAp (0) is higher than that in the above control. In addition to the affinity of HAp ceramics for cells, calcium ions slightly eluted from HAp ceramics However, this is considered to be a result of increasing the cell culture efficiency.

4). Responsiveness of mouse-derived spleen cells in medium containing IP ₆ (Reference Example 3)
Experimental Method Medium Preparation RPMI medium was used as the medium. RPMI medium was RPMI 5.20 g, penicillin 0.035 g, streptomycin 0.070 g, mercaptoethanol 1.769 μl in 500 cm ³ ultrapure water, stirred, NaHCO ₃ 0.8 g was added, pH And FBS added at 55 cm ³ was used.
Also, IP ₆ solution was added to RPMI medium 10 cm ³ within 5 vol%, so that IP ₆ concentration of 10ppm (w / v), was prepared IP ₆ containing medium.

Cultivation of mouse spleen cells One 12-week-old mouse (C57BL / 6NCrSlc) was sacrificed with diethyl ether, and the spleen was removed into RPMI medium (6 cm dish) cooled to 4 ° C. Spleen cells were taken out by sandwiching and crushing between sterilized nylon mesh sheets, and transferred to a 15 ml centrifuge tube through the nylon mesh sheet. The medium was added to the petri dish, and the cells were washed twice in the centrifuge tube. Red blood cells were removed by adding Ack Solution, bursting red blood cells for 5 minutes, adding 2 volumes of PBS, and centrifuging at 1500 rpm, 5 min, 4 ° C. twice. The IP ₆ to prepare a suspension added IP ₆ containing medium containing a concentration of 10ppm (w / v), the suspension was diluted 10-fold with trypan blue was determined the number of cells using a hemocytometer .
Suspension was prepared so that it might become 1.0 * 10 < ⁶ > cells / ml, and it seed | inoculated on 24 well plate. After culturing for 24 hours in an incubator at 37 ° C. and 5% CO ₂ , Live / Dead cell staining was performed in the same manner as described above, reacted with the antibody, and analyzed by a flow cytometer.

(Reference Example 4)
Instead of the IP ₆ containing media containing IP ₆ at a concentration of 10ppm (w / v), except that culturing mouse spleen cells with IP ₆ containing medium containing IP ₆ at a concentration of 50ppm (w / v) is The culture was carried out in the same manner as in Reference Example 3.
(Reference Example 5)
Instead of the IP ₆ containing media containing IP ₆ at a concentration of 10ppm (w / v), except that culturing mouse spleen cells with IP ₆ containing medium containing IP ₆ at a concentration of 100ppm (w / v) is The culture was carried out in the same manner as in Reference Example 3.
(Reference Example 6)
Instead of IP ₆ to IP ₆ containing media at a concentration of 10ppm (w / v), except that culturing mouse spleen cells with IP ₆ containing medium containing IP ₆ at a concentration of 150ppm (w / v) is The culture was carried out in the same manner as in Reference Example 3.
(Reference Example 7)
Instead of the IP ₆ containing media containing IP ₆ at a concentration of 10ppm (w / v), except that culturing mouse spleen cells with IP ₆ containing medium containing IP ₆ at a concentration of 300ppm (w / v) is The culture was carried out in the same manner as in Reference Example 3.
(Reference Example 8)
Instead of the IP ₆ containing media containing IP ₆ at a concentration of 10ppm (w / v), except that culturing mouse spleen cells with IP ₆ containing medium containing IP ₆ at a concentration of 500ppm (w / v) is The culture was carried out in the same manner as in Reference Example 3.
(Reference Example 9)
Instead of the IP ₆ containing media containing IP ₆ at a concentration of 10ppm (w / v), except that culturing mouse spleen cells with IP ₆ containing medium containing IP ₆ at a concentration of 1000ppm (w / v) is The culture was carried out in the same manner as in Reference Example 3.
(Reference Example 10)
Instead of the IP ₆ containing media containing IP ₆ at a concentration of 10ppm (w / v), except that culturing mouse spleen cells with IP ₆ containing medium containing IP ₆ at a concentration of 2000ppm (w / v) is The culture was carried out in the same manner as in Reference Example 3.
(Reference Example 11)
Instead of the IP ₆ containing media containing IP ₆ at a concentration of 10ppm (w / v), except that culturing mouse spleen cells with IP ₆ containing medium containing IP ₆ at a concentration of 3000ppm (w / v) is The culture was carried out in the same manner as in Reference Example 3.
(Reference Example 12)
Instead of the IP ₆ containing media containing IP ₆ at a concentration of 10ppm (w / v), except that culturing mouse spleen cells with IP ₆ containing medium containing IP ₆ at a concentration of 4000ppm (w / v) is The culture was carried out in the same manner as in Reference Example 3.
(Reference Example 13)
Instead of the IP ₆ containing media containing IP ₆ at a concentration of 10ppm (w / v), except that culturing mouse spleen cells with IP ₆ containing medium containing IP ₆ at a concentration of 5000ppm (w / v) is The culture was carried out in the same manner as in Reference Example 3.

The results are shown in FIGS. The vertical axis of the graph of FIG. 10 is the rate of increase / decrease of helper T cells after culturing on IP ₆ -HAp ceramics or in an IP _6- containing medium relative to the ratio of helper T cells in the control after 1 day of culture of the seeded cells. . 3. above. The results of the increase / decrease rate of helper T cells cultured on IP ₆ -HAp ceramics in Reference Example 2 and Examples 5 to 8 are shown in FIG. 10 (a), and Reference Examples 3 to 7 and Reference Examples 9 to 13 are shown. the IP _6% change helper T cells cultured in medium containing results shown in Figure 10 (b).
When cells were cultured in a medium containing IP ₆ , the increase / decrease rate of the cells was about 12% when the IP ₆ concentration was 3000 ppm. When cells were cultured on IP ₆ -HAp ceramics, when IP ₆ -HAp (3000) was used, the increase / decrease rate of the cells was about 14%, which was almost the same value as in the medium. _However, IP 6 -HAp (3000) is, IP ₆ concentration are the base material obtained by immersing the IP ₆ a solution of 3000 ppm, actually _IP 6 -HAp bonded to that of IP ₆ in (3000) the total amount is compared to the total amount of IP ₆ in IP ₆ containing media containing IP ₆ in 3000 ppm, it is very small.
Therefore, it can be seen that by using IP ₆ -HAp ceramics to which IP ₆ is bonded, the same effect can be obtained by using a small amount of IP ₆ as compared with the case of using a medium containing IP ₆ .

3. above. Or 4. The result of having performed Live / Dead cell dyeing | staining with respect to the spleen cell cultured in the term is shown. In the medium containing IP ₆ and the IP ₆ concentration was 10, 50, 100, 150, 300, 1000, 2000, 3000, 4000, 5000 ppm, the ratio of viable cells in the whole cells was 49 on average. 15%.
In contrast, cells cultured under the conditions of IP ₆ -HAp (0), IP ₆ -HAp (1000), IP ₆ -HAp (2000), IP ₆ -HAp (3000), and IP ₆ -HAp (50000) The ratio of living cells in the whole was 73.46% on average.
Therefore, it can be seen that the cell viability is higher on the IP ₆ -HAp ceramics than in the IP _6- containing medium, and the cells can be cultured well.

The vertical axis of the graph of FIG. 11 represents the rate of increase / decrease of killer T cells after culturing on IP ₆ -HAp ceramics or in an IP _6- containing medium relative to the ratio of killer T cells in the control after 1 day of culture of the seeded cells. . 3. above. The results of the increase / decrease rate of killer T cells cultured on IP ₆ -HAp ceramics in Reference Example 2 and Examples 5 to 8 in the section are shown in FIG. 11 (a), and the IP ₆ -containing medium in Reference Examples 3 to 13 The result of the increase / decrease rate of the killer T cells cultured in is shown in FIG.
When cells were cultured on IP ₆ -HAp ceramics, the increase / decrease rate of cells when IP ₆ -HAp (3000) was used was about 10%. However, when cells were cultured in a medium containing IP ₆ , when the IP ₆ concentration was 5000 ppm, the increase / decrease rate of the cells was remarkably reduced to about minus 15%.
This result shows that an IP ₆ concentration that is too high tends to induce cell death. Risk IP ₆ is a _IP 6 -HAp ceramic coupled to HAp, actually total amount of IP ₆ attached to _IP 6 -HAp (3000), because it is extremely small, which induces such cell death It can be seen that the cells can be cultured well.

  The configurations and combinations thereof in the embodiments described above are examples, and the addition, omission, replacement, and other modifications of the configurations can be made without departing from the spirit of the present invention. Further, the present invention is not limited by each embodiment, and is limited only by the scope of the claims.

  According to the present invention, a cell culture substrate capable of culturing immune cells and having an immunostimulatory action, a method for producing the cell culture substrate, a cell culture method using the cell culture substrate, and the cell culture A cell culture device provided with a substrate can be provided.

DESCRIPTION OF SYMBOLS 1,11 ... Cell culture substrate, 2 ... Metal compound, 12 ... Hydroxyapatite layer, 13 ... Center layer, 4 ... Inositol phosphate, 20 ... Cell culture apparatus, 25 ... Culture tank

Claims (9)

The surface even without low, including the metal compound of inositol phosphate or its salt are combined,
The cell culture substrate , wherein the metal compound is a ceramic of a calcium phosphate compound ,
Use to culture T cells in contact with outside the body . The use according to claim 1, wherein the inositol phosphate is inositol hexaphosphate. At least the surface has a metal compound to which inositol phosphate or a salt thereof is bound,
A cell culture substrate in which the metal compound is a ceramic of a calcium phosphate compound;
T cells in contact with the cell culture substrate;
A cell culture substrate with a cell.   The cell culture substrate with a cell according to claim 3, wherein the inositol phosphate is inositol hexaphosphate.   The method for producing a cell culture substrate with a cell according to claim 3 or 4, wherein a solution containing inositol phosphate or a salt thereof is contacted with a metal compound, and the T cell is contacted with the obtained cell culture substrate. .   A cell having a metal compound to which inositol phosphate or a salt thereof is bound at least on the surface, the T compound being brought into contact with a cell culture substrate, wherein the metal compound is a ceramic of a calcium phosphate compound, and the T cell is cultured. Culture method.   At least the surface has a metal compound to which inositol phosphate or a salt thereof is bound,
  A cell culture apparatus comprising a cell culture substrate in which the metal compound is a ceramic of a calcium phosphate compound, and a culture tank for containing the cell culture substrate, for bringing T cells into contact with each other and culturing them outside the body. use.   8. Use according to claim 7, wherein the inositol phosphate is inositol hexaphosphate. And cell with cell culture substrate according to 請 Motomeko 3 or 4, pre SL cell culture device comprising a culture tank for containing the cells with cell culture substrate.

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