JPWO2019225600A1 - Evaluation method of hydrogel - Google Patents
Evaluation method of hydrogel Download PDFInfo
- Publication number
- JPWO2019225600A1 JPWO2019225600A1 JP2020521250A JP2020521250A JPWO2019225600A1 JP WO2019225600 A1 JPWO2019225600 A1 JP WO2019225600A1 JP 2020521250 A JP2020521250 A JP 2020521250A JP 2020521250 A JP2020521250 A JP 2020521250A JP WO2019225600 A1 JPWO2019225600 A1 JP WO2019225600A1
- Authority
- JP
- Japan
- Prior art keywords
- hydrogel
- cells
- measurement
- evaluating
- measurement step
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
ハイドロゲル上で接着性細胞を培養し、生細胞内で発色する染色剤を用いて生細胞を染色し、顕微鏡観察により生細胞数を測定する第１測定工程と、第１測定工程後にハイドロゲルを洗浄し、顕微鏡観察により生細胞数を測定する第２測定工程と、第１測定工程における測定結果と第２測定工程における測定結果とに基づき、ハイドロゲルに対する細胞接着性を評価する接着性評価工程と、を含むハイドロゲルの評価方法を提供する。Adhesive cells are cultured on a hydrogel, the viable cells are stained with a stain that develops color in the viable cells, and the number of viable cells is measured by microscopic observation. Adhesion evaluation to evaluate cell adhesion to hydrogel based on the second measurement step of washing and measuring the number of viable cells by microscopic observation, the measurement result in the first measurement step, and the measurement result in the second measurement step. Provided are a process and a method for evaluating a hydrogel including.
Description
本発明は、ハイドロゲルの評価方法に関する。 The present invention relates to a method for evaluating a hydrogel.
近年、三次元網目構造を有する高分子であるハイドロゲルが、医療用材料として注目されている。ハイドロゲルは、上記三次元網目構造によって多量の水を保持している。 In recent years, hydrogel, which is a polymer having a three-dimensional network structure, has attracted attention as a medical material. The hydrogel retains a large amount of water due to the three-dimensional network structure described above.
ハイドロゲルとしては、プルラン、デキストラン、コラーゲン、アルギン酸塩、ポリビニルアルコール等に由来する種々のハイドロゲルが知られており、それらの機械的強度、保水性、細胞接着性、生体適合性等の特性に応じた用途がある。例えば、細胞接着性の高いハイドロゲルは、細胞の足場として再生医療等の分野で利用することができる。反対に、細胞接着性の低いハイドロゲルは、治療薬を含有する創傷被覆剤等として利用することができる。 Various hydrogels derived from pullulan, dextran, collagen, alginate, polyvinyl alcohol, etc. are known as hydrogels, and their mechanical strength, water retention, cell adhesion, biocompatibility, and other characteristics are exhibited. There are different uses. For example, a hydrogel having high cell adhesion can be used as a scaffold for cells in fields such as regenerative medicine. On the contrary, the hydrogel having low cell adhesion can be used as a wound dressing or the like containing a therapeutic agent.
例えば、特許文献１には、組織修復、組織再生等に適している細胞の足場として用いることができる架橋したプルラン及びデキストランの混合物を含むハイドロゲルが開示されている。 For example, Patent Document 1 discloses a hydrogel containing a mixture of crosslinked pullulan and dextran that can be used as a scaffold for cells suitable for tissue repair, tissue regeneration and the like.
上述した通り、近年、特性の異なる様々なハイドロゲルが開発されているため、それらの細胞接着性、生体適合性等の特性を簡便に評価し、用途に応じて最適なハイドロゲルを選択することが望まれる。 As described above, since various hydrogels having different characteristics have been developed in recent years, it is necessary to easily evaluate their characteristics such as cell adhesion and biocompatibility and select the most suitable hydrogel according to the application. Is desired.
本発明は、上記に鑑みてなされたものであり、ハイドロゲルの細胞接着性及び細胞毒性を簡便に測定することができるハイドロゲルの評価方法を提供することを目的とする。 The present invention has been made in view of the above, and an object of the present invention is to provide a method for evaluating a hydrogel, which can easily measure the cell adhesion and cytotoxicity of the hydrogel.
上記課題を解決するための具体的な手段には、以下の実施態様が含まれる。
＜１＞ハイドロゲル上で接着性細胞を培養し、生細胞内で発色する染色剤を用いて生細胞を染色し、顕微鏡観察により生細胞数を測定する第１測定工程と、該第１測定工程後に前記ハイドロゲルを洗浄し、顕微鏡観察により生細胞数を測定する第２測定工程と、前記第１測定工程における測定結果と前記第２測定工程における測定結果とに基づき、前記ハイドロゲルに対する細胞接着性を評価する接着性評価工程と、を含むハイドロゲルの評価方法。Specific means for solving the above problems include the following embodiments.
<1> A first measurement step of culturing adhesive cells on a hydrogel, staining the living cells with a stain that develops color in the living cells, and measuring the number of living cells by microscopic observation, and the first measurement. Based on the second measurement step of washing the hydrogel after the step and measuring the number of living cells by microscopic observation, the measurement result in the first measurement step, and the measurement result in the second measurement step, the cells for the hydrogel. A method for evaluating a hydrogel, which comprises an adhesiveness evaluation step for evaluating adhesiveness.
＜２＞前記第１測定工程及び前記第２測定工程では、プレート状のハイドロゲル基材に設けられた凹部内で前記接着性細胞を培養する＜１＞に記載のハイドロゲルの評価方法。 <2> The method for evaluating a hydrogel according to <1>, wherein in the first measurement step and the second measurement step, the adhesive cells are cultured in a recess provided in a plate-shaped hydrogel base material.
＜３＞前記第１測定工程における測定結果に基づき、前記ハイドロゲルの細胞毒性を評価する毒性評価工程をさらに含む＜１＞又は＜２＞に記載のハイドロゲルの評価方法。 <3> The method for evaluating a hydrogel according to <1> or <2>, further comprising a toxicity evaluation step for evaluating the cytotoxicity of the hydrogel based on the measurement result in the first measurement step.
＜４＞前記染色剤がＭＴＴである＜１＞〜＜３＞のいずれか１項に記載のハイドロゲルの評価方法。 <4> The method for evaluating a hydrogel according to any one of <1> to <3>, wherein the dyeing agent is MTT.
＜５＞ハイドロゲル上で細胞を培養し、生細胞内で発色する染色剤を用いて生細胞を染色し、顕微鏡観察により生細胞数を測定する測定工程と、前記測定工程における測定結果に基づき、前記ハイドロゲルの細胞毒性を評価する毒性評価工程と、を含むハイドロゲルの評価方法。 <5> Based on a measurement step of culturing cells on a hydrogel, staining the living cells with a staining agent that develops color in the living cells, and measuring the number of living cells by microscopic observation, and the measurement results in the measurement step. A method for evaluating a hydrogel, which comprises a toxicity evaluation step for evaluating the cytotoxicity of the hydrogel.
本発明によれば、ハイドロゲルの細胞接着性及び細胞毒性を簡便に測定することができるハイドロゲルの評価方法を提供することができる。 According to the present invention, it is possible to provide a method for evaluating a hydrogel, which can easily measure the cell adhesion and cytotoxicity of the hydrogel.
以下、本発明の実施形態について説明する。ただし、本発明は以下の実施形態に限定されるものではない。 Hereinafter, embodiments of the present invention will be described. However, the present invention is not limited to the following embodiments.
［第１実施形態］
本発明の第１実施形態に係るハイドロゲルの評価方法は、ハイドロゲル上で接着性細胞を培養し、生細胞内で発色する染色剤を用いて生細胞を染色し、顕微鏡観察により生細胞数を測定する第１測定工程と、該第１測定工程後に上記ハイドロゲルを洗浄し、顕微鏡観察により生細胞数を測定する第２測定工程と、上記第１測定工程における測定結果と上記第２測定工程における測定結果とに基づき、上記ハイドロゲルに対する細胞接着性を評価する接着性評価工程と、を含む。[First Embodiment]
In the method for evaluating a hydrogel according to the first embodiment of the present invention, adherent cells are cultured on the hydrogel, the living cells are stained with a staining agent that develops color in the living cells, and the number of living cells is observed under a microscope. The first measurement step of measuring the above, the second measurement step of washing the hydrogel after the first measurement step and measuring the number of living cells by microscopic observation, the measurement result in the first measurement step, and the second measurement. It includes an adhesion evaluation step of evaluating cell adhesion to the hydrogel based on the measurement result in the step.
評価対象となるハイドロゲルは、水を保持している三次元網目構造を有する高分子であれば特に制限されない。例えば、寒天、カラギーナン、デキストラン、プルラン、キトサン、セルロース、ゼラチン、セリシン、フィブロイン、コラーゲン、ヒアルロン酸、コンドロイチン硫酸、ポリエチレングリコール（ＰＥＧ）、ポリアクリルアミド（ＰＡＡ）、ポリビニルアルコール（ＰＶＡ）等に由来するハイドロゲルが挙げられる。また、上記高分子の誘導体に由来するハイドロゲルであってもよい。 The hydrogel to be evaluated is not particularly limited as long as it is a polymer having a three-dimensional network structure that retains water. For example, hydro derived from agar, carrageenan, dextran, pullulan, chitosan, cellulose, gelatin, sericin, fibroin, collagen, hyaluronic acid, chondroitin sulfate, polyethylene glycol (PEG), polyacrylamide (PAA), polyvinyl alcohol (PVA) and the like. Gel is mentioned. Further, it may be a hydrogel derived from the derivative of the above polymer.
ハイドロゲルの形態は特に制限されないが、マイクロプレート、シャーレ等に充填したハイドロゲルであっても、鋳型を用いて作成した凹部を有するプレート状のハイドロゲル基材であってもよい。 The form of the hydrogel is not particularly limited, and it may be a hydrogel filled in a microplate, a petri dish, or the like, or a plate-shaped hydrogel base material having recesses prepared by using a mold.
凹部を有するプレート状のハイドロゲル基材の作製方法としては、例えば、中心に凸部を有する鋳型に溶液を流し込み、凍結融解法等によりゲル化した後、ハイドロゲルを取り外し反転させることにより、中心に凹部を有するプレート状のハイドロゲル基材を作製する方法等が挙げられる。凍結融解の条件は、ＰＶＡ誘導体を例に挙げると、−２０℃で１８時間凍結し、３５℃で６時間融解する工程を１サイクルとし、これを数回行う条件としてもよい。 As a method for producing a plate-shaped hydrogel base material having a concave portion, for example, a solution is poured into a mold having a convex portion in the center, gelled by a freeze-thaw method or the like, and then the hydrogel is removed and inverted to form a center. Examples thereof include a method of producing a plate-shaped hydrogel base material having a recess in the base. Taking a PVA derivative as an example, the freeze-thaw condition may be a step of freezing at −20 ° C. for 18 hours and thawing at 35 ° C. for 6 hours as one cycle, and this may be performed several times.
ハイドロゲルに対する細胞接着性の評価に用いる細胞は、接着性細胞であれば特に制限されない。 The cells used for evaluating the cell adhesion to the hydrogel are not particularly limited as long as they are adherent cells.
接着性細胞を培養しているハイドロゲル上に、染色剤を滴下して、適切な時間インキュベートする。 The stain is added dropwise onto the hydrogel in which the adherent cells are cultured and incubated for an appropriate time.
染色剤は、生細胞内で発色する染色剤であれば特に制限されず、３−（４，５−ジメチル−２−チアゾリル）−２，５−ジフェニル−２Ｈ−テトラゾリウムブロミド（ＭＴＴ）、カルセインアセトキシメチルエステル（カルセインＡＭ）、７−イソブトキシカルボニルオキシ−３Ｈ−フェノキサジン−３−オン（ＣｙｔｏＲｅｄ）、二酢酸フルオレセイン（ＦＤＡ）、５−（６−）−カルボキシフルオレセインジアセテートスクシンイミジルエステル（ＣＦＳＥ）、２'，７'−ビス−(２−カルボキシエチル)−５−（６−）−カルボキシフルオレセイン，アセトキシメチルエステル（ＢＣＥＣＦ−ＡＭ）等が挙げられる。 The stain is not particularly limited as long as it is a stain that develops color in living cells, and is 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide (MTT), calcein acetoxy. Methyl ester (calcein AM), 7-isobutoxycarbonyloxy-3H-phenoxazine-3-one (CytoRed), fluorescein diacetate (FDA), 5- (6-) -carboxyfluorescein diacetate succinimidyl ester (CFSE) , 2', 7'-bis- (2-carboxyethyl) -5- (6-) -carboxyfluorescein, acetoxymethyl ester (BCECF-AM) and the like.
上記染色剤は、生細胞内に取り込まれると酵素によって加水分解、還元等されることにより発色する。例えば、ＭＴＴを例に挙げると、黄色の溶液であるＭＴＴが、脱水素酵素によって還元されて難溶性沈殿物である青色のホルマザン色素に変換される。上記脱水素酵素は、ミトコンドリアの電子伝達系に関連する酵素であるため、死細胞ではホルマザン色素は生成されない。 When the dyeing agent is taken up into living cells, it is hydrolyzed or reduced by an enzyme to develop a color. For example, taking MTT as an example, MTT, which is a yellow solution, is reduced by dehydrogenase and converted into a blue formazan dye, which is a poorly soluble precipitate. Since the dehydrogenase is an enzyme related to the electron transport chain of mitochondria, formazan pigment is not produced in dead cells.
ハイドロゲルは多孔質構造を有し、染色剤を吸着するため、ハイドロゲル上の細胞に染色剤を滴下すると、ハイドロゲルも細胞と同様に染色される。生細胞内で発色する染色剤を用いた場合、染色剤が上記の作用機序により生細胞内で異なる色素に変換されるため、生細胞はハイドロゲルと異なる色に染色される。一方、生細胞内で発色が起こらない染色剤を用いてハイドロゲル上の細胞を染色する場合、ハイドロゲルと細胞とが同色に染色されるため、細胞を識別することが困難となる。 Since the hydrogel has a porous structure and adsorbs the stain, when the stain is dropped on the cells on the hydrogel, the hydrogel is stained in the same manner as the cells. When a stain that develops a color in the living cell is used, the dye is converted into a different pigment in the living cell by the above-mentioned mechanism of action, so that the living cell is stained in a color different from that of the hydrogel. On the other hand, when the cells on the hydrogel are stained with a stain that does not cause color development in the living cells, the hydrogel and the cells are stained in the same color, which makes it difficult to identify the cells.
染色された生細胞数は、顕微鏡観察により測定される。測定方法については特に制限されないが、例えば、目視により生細胞数を計数してもよく、画像解析により生細胞に起因する発色部の面積を測定して生細胞数とみなしてもよい。この結果得られたハイドロゲル上の生細胞数を、第１測定工程における測定結果とする。 The number of viable cells stained is measured by microscopic observation. The measuring method is not particularly limited, but for example, the number of viable cells may be visually counted, or the area of the color-developing portion caused by the viable cells may be measured by image analysis and regarded as the number of viable cells. The number of living cells on the hydrogel obtained as a result is used as the measurement result in the first measurement step.
顕微鏡の種類は、特に制限されないが、光透過性が低い半透明又は不透明なハイドロゲルに対しては、透過型顕微鏡ではなく落射型顕微鏡を用いる。 The type of microscope is not particularly limited, but for translucent or opaque hydrogels having low light transmission, an epi-illumination microscope is used instead of a transmission microscope.
顕微鏡を用いて観察することにより、生細胞を簡便に測定することが可能となる。 By observing with a microscope, living cells can be easily measured.
第１測定工程後、ハイドロゲルを洗浄する。これにより、ハイドロゲル上に接着しなかった細胞は除去される。 After the first measurement step, the hydrogel is washed. This removes the cells that did not adhere to the hydrogel.
第１測定工程における測定方法と同様の方法により、洗浄されたハイドロゲル上の生細胞数を測定する。この結果得られたハイドロゲルに接着した生細胞数を、第２測定工程における測定結果とする。 The number of viable cells on the washed hydrogel is measured by the same method as the measurement method in the first measurement step. The number of viable cells adhered to the hydrogel obtained as a result is used as the measurement result in the second measurement step.
ハイドロゲルの細胞接着性は、第１測定工程における測定結果と第２測定工程における測定結果とに基づき評価される。具体的には、例えば、第１測定工程における測定結果に対する第２測定工程における測定結果の比を細胞接着率として算出し評価してもよい。 The cell adhesion of the hydrogel is evaluated based on the measurement result in the first measurement step and the measurement result in the second measurement step. Specifically, for example, the ratio of the measurement result in the second measurement step to the measurement result in the first measurement step may be calculated and evaluated as the cell adhesion ratio.
また、本実施形態に係るハイドロゲルの評価方法は、第１測定工程における測定結果に基づき、ハイドロゲルの細胞毒性を評価する工程を含んでもよい。例えば、予めハイドロゲルに播種される細胞に含まれる生細胞数を確認し、第１測定工程にて得られた生細胞数との差から、培養中にハイドロゲルの影響を受けて死滅した細胞数を算出することができる。上記の細胞毒性を評価する工程を含む場合、一連の工程において、ハイドロゲルの細胞接着性と細胞毒性とを評価することができる。 Further, the method for evaluating the hydrogel according to the present embodiment may include a step of evaluating the cytotoxicity of the hydrogel based on the measurement result in the first measurement step. For example, the number of viable cells contained in the cells seeded in the hydrogel is confirmed in advance, and the difference from the number of viable cells obtained in the first measurement step indicates that the cells died under the influence of the hydrogel during the culture. The number can be calculated. When the above-mentioned step of evaluating cytotoxicity is included, the cell adhesion and cytotoxicity of the hydrogel can be evaluated in a series of steps.
［第２実施形態］
本発明の第２実施形態に係るハイドロゲルの評価方法は、ハイドロゲル上で細胞を培養し、生細胞内で発色する染色剤を用いて生細胞を染色し、顕微鏡観察により生細胞数を測定する測定工程と、該測定工程における測定結果に基づき、上記ハイドロゲルの細胞毒性を評価する毒性評価工程とを含む。[Second Embodiment]
In the method for evaluating a hydrogel according to a second embodiment of the present invention, cells are cultured on the hydrogel, the living cells are stained with a staining agent that develops color in the living cells, and the number of living cells is measured by microscopic observation. The measurement step includes a toxicity evaluation step of evaluating the cytotoxicity of the hydrogel based on the measurement result in the measurement step.
本実施形態に係る評価対象となるハイドロゲル、染色剤、及び顕微鏡観察による生細胞数の測定方法については、第１実施形態と同様であるため、その説明を省略する。 Since the hydrogel, the dyeing agent, and the method for measuring the number of living cells by microscopic observation according to the present embodiment are the same as those in the first embodiment, the description thereof will be omitted.
ハイドロゲルに対する細胞毒性の評価に用いる細胞は、特に制限されず、接着性細胞であっても、浮遊性細胞であってもよい。 The cells used for evaluating the cytotoxicity to the hydrogel are not particularly limited, and may be adhesive cells or floating cells.
細胞培養及び染色工程では、予め生細胞数を測定した細胞懸濁液をハイドロゲル上に播種し、インキュベートし、染色剤を滴下した後、適切な時間インキュベートする。 In the cell culture and staining steps, a cell suspension in which the number of viable cells has been measured in advance is seeded on a hydrogel, incubated, a stain is added dropwise, and then incubated for an appropriate time.
染色された生細胞数を顕微鏡観察により測定し、この測定結果と最初に播種した生細胞数とに基づき、ハイドロゲルの細胞毒性を評価する。具体的には、例えば、最初に播種した生細胞数に対する上記顕微鏡観察による測定結果の比を細胞毒性率として算出し評価してもよい。 The number of stained viable cells is measured by microscopic observation, and the cytotoxicity of the hydrogel is evaluated based on the measurement result and the number of viable cells initially seeded. Specifically, for example, the ratio of the measurement result by the above-mentioned microscopic observation to the number of viable cells first seeded may be calculated and evaluated as the cytotoxicity rate.
第１実施形態及び第２実施形態に係るハイドロゲルの評価方法によれば、ハイドロゲルの細胞接着性及び細胞毒性を簡便に測定することができる。これにより、用途に応じて有効なハイドロゲルを選別することができる。 According to the hydrogel evaluation methods according to the first embodiment and the second embodiment, the cell adhesion and cytotoxicity of the hydrogel can be easily measured. This makes it possible to select an effective hydrogel according to the application.
以下、本発明の実施例を説明するが、本発明の範囲はこれらの実施例に限定されるものではない。 Hereinafter, examples of the present invention will be described, but the scope of the present invention is not limited to these examples.
［実施例１］
実施例１では、以下の方法により、ハイドロゲルの細胞接着性試験及び細胞毒性試験を行った。[Example 1]
In Example 1, the cell adhesion test and the cytotoxicity test of the hydrogel were carried out by the following methods.
＜ハイドロゲル基材の調製＞
評価対象となるハイドロゲル基材は、以下のように作製した。
まず、ポリビニルアルコール（ＰＶＡ）の誘導体であるＥＸＣＥＶＡＬ（登録商標、クラレ社製）に超純水を加え、ＥＸＣＥＶＡＬの濃度が１０ｗ／ｖ％の溶液を調製した。次いで、１８０℃で１時間、乾熱滅菌した金属片（直径２０ｍｍ、厚さ１０ｍｍ）をステンレス製シャーレ（直径４０ｍｍ、厚さ１５ｍｍ）の中心に設置した。そして、上記１０ｗ／ｖ％ＥＸＣＥＶＡＬ溶液を、上記ステンレス製シャーレ上に充填し、凍結融解法によりゲル化した。その後、ゲル化した１０ｗ／ｖ％ＥＸＣＥＶＡＬのハイドロゲルを上記ステンレス製シャーレから取り外し反転させて、凹部を有するプレート状のハイドロゲル基材を作製した。なお、凍結融解は、−２０℃で１８時間凍結し、３５℃で６時間融解する工程を１サイクル行った。<Preparation of hydrogel base material>
The hydrogel base material to be evaluated was prepared as follows.
First, ultrapure water was added to EXCEVAL (registered trademark, manufactured by Kuraray Co., Ltd.), which is a derivative of polyvinyl alcohol (PVA), to prepare a solution having an EXCEVAL concentration of 10 w / v%. Next, a metal piece (diameter 20 mm, thickness 10 mm) sterilized by dry heat at 180 ° C. for 1 hour was placed in the center of a stainless steel petri dish (diameter 40 mm, thickness 15 mm). Then, the 10 w / v% EXCEVAL solution was filled on the stainless steel petri dish and gelled by the freeze-thaw method. Then, the gelled 10 w / v% EXCEVAL hydrogel was removed from the stainless steel petri dish and inverted to prepare a plate-shaped hydrogel base material having recesses. For freezing and thawing, one cycle of freezing at −20 ° C. for 18 hours and thawing at 35 ° C. for 6 hours was performed.
＜細胞接着性試験＞
まず、調製した１０ｗ／ｖ％ＥＸＣＥＶＡＬのハイドロゲル基材の凹部に、ＨａＣａＴ細胞を１００００細胞／ｃｍ２となるよう播種して２４時間インキュベートした後、ＭＴＴ試薬を滴下して２時間インキュベートした。次いで、落射型顕微鏡を用いて、上記ハイドロゲル基材の凹部を観察して、ＭＴＴ試薬により発色した生細胞数を測定した。そして、上記ハイドロゲル基材を振とう機を用いてリン酸緩衝液（ＰＢＳ）で１５分間振とう洗浄した。その後、再び落射型顕微鏡にて上記ハイドロゲル基材の凹部を観察して、生細胞数を測定した。<Cell adhesion test>
First, HaCaT cells were seeded in the recesses of the prepared hydrogel substrate of 10 w / v% EXCEVAL so as to be 10000 cells / cm 2 and incubated for 24 hours, and then the MTT reagent was added dropwise and incubated for 2 hours. Next, using an epi-illumination microscope, the recesses of the hydrogel substrate were observed, and the number of viable cells colored by the MTT reagent was measured. Then, the hydrogel substrate was washed with a phosphate buffer solution (PBS) for 15 minutes using a shaker. Then, the concave portion of the hydrogel base material was observed again with an epi-illumination microscope, and the number of living cells was measured.
図１ＡはＰＢＳで洗浄前の上記ハイドロゲル基材の顕微鏡画像、図１ＢはＰＢＳで洗浄後の上記ハイドロゲル基材の顕微鏡画像である。図１Ａから、上記ハイドロゲル上での細胞の生存が確認された。そして、図１Ｂから、上記ハイドロゲル上の細胞数は明らかに減少し、ほとんどの細胞が洗い流されたことが確認された。このことから、１０ｗ／ｖ％ＥＸＣＥＶＡＬハイドロゲル上に生存していたほとんどの細胞は接着していなかったと考えられる。 FIG. 1A is a microscopic image of the hydrogel base material before washing with PBS, and FIG. 1B is a microscopic image of the hydrogel base material after washing with PBS. From FIG. 1A, the survival of cells on the hydrogel was confirmed. Then, from FIG. 1B, it was confirmed that the number of cells on the hydrogel was clearly reduced and most of the cells were washed away. From this, it is considered that most of the cells alive on the 10 w / v% EXCEVAL hydrogel were not adhered.
＜細胞毒性試験＞
細胞接着性試験と同様に、調製した１０ｗ／ｖ％ＥＸＣＥＶＡＬのハイドロゲル基材の凹部に、ＨａＣａＴ細胞を１００００細胞／ｃｍ２となるよう播種して２４時間インキュベートした後、ＭＴＴ試薬を滴下して２時間インキュベートした。次いで、落射型顕微鏡を用いて、上記ハイドロゲル基材の凹部を観察して、ＭＴＴ試薬により発色した生細胞数を測定した。細胞の生存が確認されたことから、細胞毒性はないと判断された。<Cytotoxicity test>
Similar to the cell adhesion test, HaCaT cells were seeded in the recesses of the prepared hydrogel substrate of 10 w / v% EXCEVAL so as to be 10000 cells / cm 2 and incubated for 24 hours, and then the MTT reagent was added dropwise. Incubated for 2 hours. Next, using an epi-illumination microscope, the recesses of the hydrogel substrate were observed, and the number of viable cells colored by the MTT reagent was measured. Since cell survival was confirmed, it was judged that there was no cytotoxicity.
［比較例１］
比較例１では、以下の方法により、ハイドロゲル上の細胞数の測定を行った。[Comparative Example 1]
In Comparative Example 1, the number of cells on the hydrogel was measured by the following method.
＜ハイドロゲルの調製＞
評価対象となるハイドロゲルを以下のように調製した。
まず、ＥＸＣＥＶＡＬに超純水を加え、ＥＸＣＥＶＡＬの濃度が２．５ｗ／ｖ％、５．０ｗ／ｖ％、７．５ｗ／ｖ％、及び１０．０ｗ／ｖ％の溶液を調製し、これら全ての濃度の溶液が異なるウェルに充填された２４ウェルプレートを４枚準備した。また、ＰＶＡであるＰＯＶＡＬ（クラレ社製）に超純水を加え、２．５ｗ／ｖ％、５．０ｗ／ｖ％、７．５ｗ／ｖ％、及び１０．０ｗ／ｖ％の溶液を調製し、これら全ての濃度の溶液が異なるウェルに充填された２４ウェルプレートを４枚準備した。
次いで、上記８枚の２４ウェルプレートに充填された溶液を、以下の４条件で凍結融解法によりゲル化した。凍結融解条件は、−２０℃で１８時間凍結し、３５℃で６時間融解する工程を１サイクル、２サイクル、３サイクル、及び４サイクルとした。そして、凍結融解したウェル内のハイドロゲルをＰＢＳで洗浄後、ＤＭＥＭ培地を加え２４時間静置した。<Preparation of hydrogel>
The hydrogel to be evaluated was prepared as follows.
First, ultrapure water was added to EXCEVAL to prepare solutions having EXCEVAL concentrations of 2.5 w / v%, 5.0 w / v%, 7.5 w / v%, and 10.0 w / v%, all of which were prepared. Four 24-well plates were prepared in which different wells were filled with solutions of the same concentration. Further, ultrapure water is added to POVAL (manufactured by Kuraray) which is PVA to prepare solutions of 2.5 w / v%, 5.0 w / v%, 7.5 w / v%, and 10.0 w / v%. Then, four 24-well plates were prepared in which solutions of all these concentrations were filled in different wells.
Next, the solution filled in the above eight 24-well plates was gelled by the freeze-thaw method under the following four conditions. As the freeze-thaw conditions, the steps of freezing at −20 ° C. for 18 hours and thawing at 35 ° C. for 6 hours were defined as 1 cycle, 2 cycles, 3 cycles, and 4 cycles. Then, the hydrogel in the freeze-thawed well was washed with PBS, DMEM medium was added, and the mixture was allowed to stand for 24 hours.
＜ハイドロゲル上の細胞数の測定＞
各条件で調製したハイドロゲルが充填されたウェル内に、ＨａＣａＴ細胞を５０００細胞／ｃｍ２で播種して２４時間インキュベートした後、ＤＭＥＭ培地を取り除き、４％パラホルムアルデヒド・リン酸緩衝液を用いて１０分間、細胞の固定処理を行った。そして、０．５％クリスタルバイオレットを用いて１５分間、細胞を染色した。その後、超純水でクリスタルバイオレットを洗い流し、落射型顕微鏡を用いて、ハイドロゲル上の細胞数を測定した。<Measurement of cell number on hydrogel>
HaCaT cells were seeded at 5000 cells / cm 2 in wells filled with hydrogels prepared under each condition and incubated for 24 hours, then the DMEM medium was removed, and 4% paraformaldehyde / phosphate buffer was used. The cells were fixed for 10 minutes. The cells were then stained with 0.5% crystal violet for 15 minutes. Then, the crystal violet was rinsed with ultrapure water, and the number of cells on the hydrogel was measured using an epi-illumination microscope.
全条件で調製したハイドロゲルにおいて、クリスタルバイオレットにより細胞が青紫色に染色されたが、ハイドロゲルも染色剤が吸着することにより青紫色に染色された。このため、ハイドロゲル上の細胞を識別できず、細胞数を測定することができなかった。そこで、２４ウェルプレートからハイドロゲルを取り出し還流洗浄した。ハイドロゲル内のクリスタルバイオレットは除去することができたが、細胞も脱色されたため、細胞数を測定できなかった。 In the hydrogel prepared under all conditions, the cells were stained bluish purple with crystal violet, but the hydrogel was also stained bluish purple due to the adsorption of the stain agent. Therefore, the cells on the hydrogel could not be identified and the number of cells could not be measured. Therefore, the hydrogel was taken out from the 24-well plate and washed with reflux. Crystal violet in the hydrogel could be removed, but the cells were also bleached and the number of cells could not be measured.
Claims (5)
前記第１測定工程後に前記ハイドロゲルを洗浄し、顕微鏡観察により生細胞数を測定する第２測定工程と、
前記第１測定工程における測定結果と前記第２測定工程における測定結果とに基づき、前記ハイドロゲルに対する細胞接着性を評価する接着性評価工程と、
を含むハイドロゲルの評価方法。The first measurement step of culturing adherent cells on a hydrogel, staining the living cells with a staining agent that develops color in the living cells, and measuring the number of living cells by microscopic observation.
After the first measurement step, the hydrogel is washed and the number of living cells is measured by microscopic observation.
Based on the measurement result in the first measurement step and the measurement result in the second measurement step, an adhesion evaluation step for evaluating cell adhesion to the hydrogel, and an adhesion evaluation step.
Evaluation method of hydrogel including.
前記測定工程における測定結果に基づき、前記ハイドロゲルの細胞毒性を評価する毒性評価工程と、
を含むハイドロゲルの評価方法。A measurement process in which cells are cultured on a hydrogel, the living cells are stained with a stain that develops color in the living cells, and the number of living cells is measured by microscopic observation.
A toxicity evaluation step for evaluating the cytotoxicity of the hydrogel based on the measurement results in the measurement step, and a toxicity evaluation step.
Evaluation method of hydrogel including.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2018098312 | 2018-05-22 | ||
| JP2018098312 | 2018-05-22 | ||
| PCT/JP2019/020099 WO2019225600A1 (en) | 2018-05-22 | 2019-05-21 | Method for evaluating hydrogel |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPWO2019225600A1 true JPWO2019225600A1 (en) | 2021-05-27 |
Family
ID=68616133
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2020521250A Pending JPWO2019225600A1 (en) | 2018-05-22 | 2019-05-21 | Evaluation method of hydrogel |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JPWO2019225600A1 (en) |
| WO (1) | WO2019225600A1 (en) |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003074099A1 (en) * | 2002-03-06 | 2003-09-12 | Japan Tissue Engineering Co.,Ltd | Base material for tissue regeneration, transplantation material and process for producing the same |
| WO2005118809A2 (en) * | 2004-04-29 | 2005-12-15 | The University Of North Carolina At Chapel Hill | Methods and compositions for enhancing cell adhesion properties |
| PL2455104T3 (en) * | 2010-11-19 | 2013-12-31 | Univ Freiburg | Bio-functionalized stimulus-responsive dissolvable PEG-hydrogels |
-
2019
- 2019-05-21 JP JP2020521250A patent/JPWO2019225600A1/en active Pending
- 2019-05-21 WO PCT/JP2019/020099 patent/WO2019225600A1/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| WO2019225600A1 (en) | 2019-11-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sakai et al. | Differentiation potential of human adipose stem cells bioprinted with hyaluronic acid/gelatin‐based bioink through microextrusion and visible light‐initiated crosslinking | |
| Katsen-Globa et al. | Towards ready-to-use 3-D scaffolds for regenerative medicine: adhesion-based cryopreservation of human mesenchymal stem cells attached and spread within alginate–gelatin cryogel scaffolds | |
| Bergonzi et al. | Study of 3D-printed chitosan scaffold features after different post-printing gelation processes | |
| CN105295073B (en) | A kind of high-flexibility amphoteric ion hydrogel preparation method | |
| AU2020103459A4 (en) | Preparation method and application of fluorescent hydrogel dressing | |
| CN106750377A (en) | Hydrogel, the composition for preparing hydrogel and hydrogel preparation method | |
| Criado-Gonzalez et al. | Enzyme assisted peptide self-assemblies trigger cell adhesion in high density oxime based host gels | |
| Lozinsky et al. | Cryostructuring of polymer systems. 47. Preparation of wide porous gelatin-based cryostructurates in sterilizing organic media and assessment of the suitability of thus formed matrices as spongy scaffolds for 3D cell culturing | |
| Zhang et al. | Mass-production of fluorescent chitosan/graphene oxide hybrid microspheres for in vitro 3D expansion of human umbilical cord mesenchymal stem cells | |
| Lai et al. | Investigation of overrun-processed porous hyaluronic acid carriers in corneal endothelial tissue engineering | |
| Gao et al. | Preparation and properties of a chitosan-based carrier of corneal endothelial cells | |
| Bidarra et al. | 3D culture of mesenchymal stem cells in alginate hydrogels | |
| US8993322B2 (en) | Methods and kits for cell release | |
| Khan et al. | Synthesis and characterization of thiol‐acrylate hydrogels using a base‐catalyzed Michael addition for 3D cell culture applications | |
| Lai | Influence of solvent composition on the performance of carbodiimide cross-linked gelatin carriers for retinal sheet delivery | |
| Hamilton et al. | Development of 3D hydrogel culture systems with on‐demand cell separation | |
| Gan et al. | GelMA/κ-carrageenan double-network hydrogels with superior mechanics and biocompatibility | |
| CN110740707B (en) | Biopsy phantom device, blood vessel wall phantom device, and method for evaluating test substance | |
| Villa et al. | P (NIPAAM–co‐HEMA) thermoresponsive hydrogels: an alternative approach for muscle cell sheet engineering | |
| JPWO2019225600A1 (en) | Evaluation method of hydrogel | |
| Tang et al. | The effects of thermoresponsive microgel density on cell adhesion, proliferation, and detachment | |
| Zhuang et al. | Thermo-responsive poly (N-isopropylacrylamide)-grafted hollow fiber membranes for osteoblasts culture and non-invasive harvest | |
| Sher et al. | Compartmentalized bioencapsulated liquefied 3D macro-construct by perfusion-based layer-by-layer technique | |
| Lai | Effect of chemical composition on corneal cellular response to photopolymerized materials comprising 2-hydroxyethyl methacrylate and acrylic acid | |
| US20060199172A1 (en) | Laminated cell culture containing hepatocytes and method for detecting endocrine-like substance |