JPH01229048A - Production of gel - Google Patents
Production of gelInfo
- Publication number
- JPH01229048A JPH01229048A JP5383488A JP5383488A JPH01229048A JP H01229048 A JPH01229048 A JP H01229048A JP 5383488 A JP5383488 A JP 5383488A JP 5383488 A JP5383488 A JP 5383488A JP H01229048 A JPH01229048 A JP H01229048A
- Authority
- JP
- Japan
- Prior art keywords
- gel
- solution
- sodium alginate
- cells
- animal cells
- 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.)
- Granted
Links
- 238000004519 manufacturing process Methods 0.000 title claims description 5
- 239000000499 gel Substances 0.000 claims abstract description 32
- 210000004027 cell Anatomy 0.000 claims abstract description 20
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000000661 sodium alginate Substances 0.000 claims abstract description 17
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 17
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 17
- 210000004102 animal cell Anatomy 0.000 claims abstract description 15
- 239000002738 chelating agent Substances 0.000 claims abstract description 10
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 229910001424 calcium ion Inorganic materials 0.000 claims abstract description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 5
- 229920002472 Starch Polymers 0.000 claims abstract description 5
- 239000011324 bead Substances 0.000 claims abstract description 5
- 239000010419 fine particle Substances 0.000 claims abstract description 5
- 239000011521 glass Substances 0.000 claims abstract description 5
- 239000008107 starch Substances 0.000 claims abstract description 5
- 235000019698 starch Nutrition 0.000 claims abstract description 5
- 238000000465 moulding Methods 0.000 claims abstract description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 3
- 239000000741 silica gel Substances 0.000 claims abstract description 3
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 3
- 210000002257 embryonic structure Anatomy 0.000 claims description 4
- 210000005061 intracellular organelle Anatomy 0.000 claims description 4
- 230000000392 somatic effect Effects 0.000 claims description 4
- 150000002500 ions Chemical class 0.000 claims 1
- 239000011347 resin Substances 0.000 claims 1
- 229920005989 resin Polymers 0.000 claims 1
- 239000000126 substance Substances 0.000 abstract description 6
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 abstract description 4
- 239000000203 mixture Substances 0.000 abstract description 3
- 239000003456 ion exchange resin Substances 0.000 abstract description 2
- 229920003303 ion-exchange polymer Polymers 0.000 abstract description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 abstract description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 abstract description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 abstract 1
- 229910052799 carbon Inorganic materials 0.000 abstract 1
- 210000001161 mammalian embryo Anatomy 0.000 abstract 1
- 238000002156 mixing Methods 0.000 abstract 1
- 210000000056 organ Anatomy 0.000 abstract 1
- 235000013311 vegetables Nutrition 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 12
- 210000004408 hybridoma Anatomy 0.000 description 5
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 239000013543 active substance Substances 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 206010020649 Hyperkeratosis Diseases 0.000 description 3
- AEMOLEFTQBMNLQ-UHFFFAOYSA-N beta-D-galactopyranuronic acid Natural products OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 230000000704 physical effect Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 239000000648 calcium alginate Substances 0.000 description 2
- 235000010410 calcium alginate Nutrition 0.000 description 2
- 229960002681 calcium alginate Drugs 0.000 description 2
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 description 2
- AEMOLEFTQBMNLQ-YBSDWZGDSA-N d-mannuronic acid Chemical compound O[C@@H]1O[C@@H](C(O)=O)[C@H](O)[C@@H](O)[C@H]1O AEMOLEFTQBMNLQ-YBSDWZGDSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000003349 gelling agent Substances 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 239000005457 ice water Substances 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- UOFGSWVZMUXXIY-UHFFFAOYSA-N 1,5-Diphenyl-3-thiocarbazone Chemical compound C=1C=CC=CC=1N=NC(=S)NNC1=CC=CC=C1 UOFGSWVZMUXXIY-UHFFFAOYSA-N 0.000 description 1
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- AEMOLEFTQBMNLQ-BZINKQHNSA-N D-Guluronic Acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@@H](O)[C@H]1O AEMOLEFTQBMNLQ-BZINKQHNSA-N 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- IAJILQKETJEXLJ-SQOUGZDYSA-N L-guluronic acid Chemical compound O=C[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O IAJILQKETJEXLJ-SQOUGZDYSA-N 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- 238000005266 casting Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- -1 dimethyl oxime Chemical class 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 210000001589 microsome Anatomy 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- VIKNJXKGJWUCNN-XGXHKTLJSA-N norethisterone Chemical compound O=C1CC[C@@H]2[C@H]3CC[C@](C)([C@](CC4)(O)C#C)[C@@H]4[C@@H]3CCC2=C1 VIKNJXKGJWUCNN-XGXHKTLJSA-N 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- MCJGNVYPOGVAJF-UHFFFAOYSA-N quinolin-8-ol Chemical compound C1=CN=C2C(O)=CC=CC2=C1 MCJGNVYPOGVAJF-UHFFFAOYSA-N 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- GCLGEJMYGQKIIW-UHFFFAOYSA-H sodium hexametaphosphate Chemical compound [Na]OP1(=O)OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])O1 GCLGEJMYGQKIIW-UHFFFAOYSA-H 0.000 description 1
- 235000019982 sodium hexametaphosphate Nutrition 0.000 description 1
- QPILZZVXGUNELN-UHFFFAOYSA-N sodium;4-amino-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound [Na+].OS(=O)(=O)C1=CC(O)=C2C(N)=CC(S(O)(=O)=O)=CC2=C1 QPILZZVXGUNELN-UHFFFAOYSA-N 0.000 description 1
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Manufacture Of Porous Articles, And Recovery And Treatment Of Waste Products (AREA)
- Compositions Of Macromolecular Compounds (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、アルギン酸カルシウムのゲルを製造する方法
に関するものである。DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for producing a calcium alginate gel.
更に詳細には、本発明は、ゲル内に多数の空ゲキを有す
るアルギン酸カルシウムのゲルを製造する方法に関する
ものである。More particularly, the present invention relates to a method for producing a calcium alginate gel having a large number of voids within the gel.
本発明によって製造されたゲルはゲル内に多数の空ゲキ
を有し、ここに動物細胞等の生理活性体を添加した場合
には、生理活性体が大量に増殖することができるために
、動物細胞等が生産する物質を大量に生産させることが
容易となり、発酵工業界、製薬界等に寄与するところ大
なるものがある。The gel produced according to the present invention has a large number of empty cells within the gel, and when bioactive substances such as animal cells are added thereto, the bioactive substances can proliferate in large quantities. It will be easier to mass-produce substances produced by cells, etc., and this will greatly contribute to the fermentation industry, pharmaceutical industry, etc.
（従来技術）
一般に動物細胞、植物細胞その他これらに関連した生理
活性体などを固定化するには、これらの１種もしくは２
種以上をアルギン酸ナトリウムなどのゲル化能を有する
物質の水溶液に懸濁し、Ｃａ”４などのゲル化剤中に滴
下してゲル化して、固定化が行なわれている。(Prior art) Generally, in order to immobilize animal cells, plant cells, and other physiologically active substances related to these, one or two of these are used.
Immobilization is carried out by suspending the seeds in an aqueous solution of a substance having gelling ability such as sodium alginate, and dropping the suspension into a gelling agent such as Ca''4 to form a gel.
（発明が解決しようとする問題点）
従来技術によって、動物細胞等を固定化する場合、自由
に増殖するだけ多数の空ゲキが与えられることはなかっ
た。適当な空ゲキがないために一定の許容範囲まで増殖
すると、動物細胞等は増殖を停止してしまって、培養生
産物を生産することもなくなってしまうのであった。(Problems to be Solved by the Invention) When animal cells and the like are immobilized using conventional techniques, they are not provided with enough cells to proliferate freely. Due to the lack of adequate energy, animal cells, etc., once they proliferate to a certain permissible range, stop proliferating and no longer produce any cultured products.
（問題点を解決するための手段）
本発明者らは、ゲルの中に多数の空ゲキを作ることがで
きれば、動物細胞等を固定化して培養する際、著しるし
く物質生産性を高めることができるとの考えから鋭意研
究したところ、特定の物性をそなえたアルギン酸ナトリ
ウムを使用すれば、固定化したとき自然に多数の空ゲキ
を形成させることができることを知ったのである。(Means for Solving the Problems) The present inventors believe that if a large number of empty cells can be created in a gel, it will significantly increase material productivity when immobilizing and culturing animal cells, etc. After conducting intensive research with the idea that this could be done, he discovered that by using sodium alginate, which has specific physical properties, it is possible to spontaneously form a large number of air bubbles when immobilized.
本発明は、この知見から完成されたもので、Ｍ／Ｇ比が
０．２−１．５で、１％溶液で１０〜１５００ｃｐ （
２５℃における）であるアルギン酸ナトリウムの００１
〜２％の溶液に、キレート剤及び動物細胞、細胞内小器
官、植物細胞、植物の不定胚、デンプン、ガラスビーズ
、活性炭、シリカゲル、イオン交換樹脂などの微粒子の
１種以上を添加したものをカルシウムイオンで適宜形状
に成型することを特徴とする空ゲキを有するゲルの製造
方法である。The present invention was completed based on this knowledge, and has an M/G ratio of 0.2-1.5 and a 1% solution of 10-1500 cp (
001 of sodium alginate) at 25°C
~2% solution to which one or more of a chelating agent and particulates such as animal cells, intracellular organelles, plant cells, plant somatic embryos, starch, glass beads, activated carbon, silica gel, and ion exchange resins are added. This is a method for producing a gel having an air bubble, which is characterized by molding it into an appropriate shape using calcium ions.
微粒子の大きさとしては０．１〜２００μｌ程度がよい
。The size of the fine particles is preferably about 0.1 to 200 μl.
本発明の空ゲキとは、ゲル中に微粒子が点在しており、
その微粒子からゲルの中心に向って空ゲキができている
ものであり、空ゲキの中は、水や培地などの液体が充満
しているものである。The dry gel of the present invention has fine particles scattered in the gel,
An air bubble is formed from the fine particles toward the center of the gel, and the air bubble is filled with liquid such as water or a culture medium.
本発明においては、特定の物性をそなえたアルギン酸ナ
トリウムが使用される。In the present invention, sodium alginate with specific physical properties is used.
一般に、アルギン酸はＤ−マンヌロン酸（Ｍ）とＬ−グ
ルロン酸（Ｇ）とから構成されていて、両者の成分比（
Ｍ／Ｇ比）が物性に大きな影響を与えている。Generally, alginic acid is composed of D-mannuronic acid (M) and L-guluronic acid (G), and the ratio of the two components is (
M/G ratio) has a great influence on physical properties.
そして、Ｍのみからなる部分、Ｇのみからなる部分、Ｍ
とＧが混在する部分などがあるがゲル化剤の金属イオン
と結合して強固なゲルを形成するのは主としてＧブロッ
クの働きによることが明らかとなっている。Then, a part consisting only of M, a part consisting only of G, M
Although there are parts where G and G are mixed, it is clear that it is mainly the function of the G block that forms a strong gel by combining with the metal ions of the gelling agent.
本発明では、アルギン酸を構成するＤ−マンヌロン酸（
Ｍ）とし−グルロン酸（Ｇ）の比、すなわちＭ／Ｇ比が
０．２〜１．５、好ましくは０．５〜１．２のものを選
んで用いる。In the present invention, D-mannuronic acid (
M) Those having a ratio of ash and guluronic acid (G), that is, an M/G ratio of 0.2 to 1.5, preferably 0.5 to 1.2, are selected and used.
また、本発明に用いるアルギン酸ナトリウムは１％溶液
で、２５℃で測定した粘度が１０〜１５００ｃｐ、好ま
しくは５０〜５００ｃｐのものでなければならない。Furthermore, the sodium alginate used in the present invention is a 1% solution and must have a viscosity of 10 to 1500 cp, preferably 50 to 500 cp, as measured at 25°C.
また、成型時のアルギン酸ナトリウムの溶液の濃度は０
．１〜２％、好ましくは０．５〜１．０％でなければな
らない。Also, the concentration of sodium alginate solution during molding is 0.
．． It should be between 1 and 2%, preferably between 0.5 and 1.0%.
以上のＭ／Ｇ比、粘度及び溶液の濃度すべてが上記特定
の範囲にあるとき、キレート剤及び動物細胞等を添加混
合して、カルシウムイオンによって成型して、はじめて
ゲル内に多数の空ゲキを形成させることができる。多数
の空ゲキを形成させるための条件は多くの実験を重ねて
確認されたもので、これらの条件をはずれれば多数の空
ゲキは形成されないことも実験によって確認されたので
ある。When the above M/G ratio, viscosity, and concentration of the solution are all within the above specified ranges, chelating agents, animal cells, etc. are added and mixed, and the gel is molded by calcium ions to form a large number of empty cells in the gel. can be formed. The conditions for forming a large number of air bubbles have been confirmed through many experiments, and it has also been confirmed through experiments that if these conditions are violated, large numbers of air bubbles will not form.
キレート剤は多数の空ゲキの形成を促進させるために添
加されるが、キレート剤の種類の例示と添加量は大約次
の通りである。A chelating agent is added to promote the formation of a large number of voids, and examples of the types of chelating agents and amounts added are roughly as follows.
重炭酸ナトリウム０．００１〜０．５％、好ましくは０
．０１〜０．１％。Sodium bicarbonate 0.001-0.5%, preferably 0
．． 01-0.1%.
ヘキサメタン酸Ｎａ ０．０００１〜０．３％、好まし
くは０．０００３〜０．０３％。Na hexamethanate 0.0001-0.3%, preferably 0.0003-0.03%.
Ｅ Ｄ Ｔ Ａ ２Ｎａ Ｏ，０００１〜２．０％、好
ましくは０．０００２〜０．０２％。EDT A 2Na O, 0001-2.0%, preferably 0.0002-0.02%.
りん酸塩（Ｎａ、に） ０．００１〜０．１％、好まし
くは０．０１〜０．０２゜
クエン酸塩（Ｎａ、 Ｋ） ０．０ＯＬ”０．１％、好
ましくは０．０１〜０．０２゜
シュウ酸０．００１〜０．１％、
好ましくはｏ、ｏｏｓ〜０．０２゜
その他のキレート剤として、硫酸、炭酸、リン酸、など
の無機酸、乳酸、クエン酸などの有機酸、縮合リン酸塩
、ジメチルオキシム、オキシン、ジチゾンなどの化合物
がある。Phosphate (Na, Ni) 0.001-0.1%, preferably 0.01-0.02°Citrate (Na, K) 0.0OL"0.1%, preferably 0.01-0.02° 0.02゜oxalic acid 0.001 to 0.1%, preferably o, oos ~ 0.02゜Other chelating agents include inorganic acids such as sulfuric acid, carbonic acid, and phosphoric acid, and organic acids such as lactic acid and citric acid. Compounds include acids, condensed phosphates, dimethyl oxime, oxine, and dithizone.
固定化する生理活性体としては、動物細胞、細胞内の小
器官、植物細胞、植物の不定胚がある。Physiologically active substances to be immobilized include animal cells, intracellular organelles, plant cells, and somatic embryos of plants.
動物細胞としては、各種生理活性物質を生産する細胞株
、抗体を生産するハイブリドーマなどがあり、単細胞で
もよく、細胞集合体でもよい、細胞内小器官としてはミ
トコンドリア、ミクロソームなどがある。また、植物細
胞としては各種物質を生産する細胞やカルスがあり、植
物の不定胚としてはカルス細胞、プロトプラスト、朽、
花粉、珠心、子房、胚珠などから誘導したものがある。Animal cells include cell lines that produce various physiologically active substances, hybridomas that produce antibodies, etc., and may be single cells or cell aggregates, and intracellular organelles include mitochondria, microsomes, etc. In addition, plant cells include cells that produce various substances and callus, and somatic embryos of plants include callus cells, protoplasts, decay, and callus.
There are those derived from pollen, nucule, ovary, ovule, etc.
キレート剤動物細胞等はアルギン酸ナトリウム溶液に添
加され、混合してカルシウムイオンによって適宜形状に
成型される。使用する液としては０．０２５〜１．０モ
ル程度の塩化カルシウム溶液などがよい。A chelating agent such as animal cells is added to a sodium alginate solution, mixed and molded into an appropriate shape by calcium ions. The liquid used is preferably a calcium chloride solution of about 0.025 to 1.0 mol.
１、 注射器やピペット等のノズルからカルシウムイオ
ンを含有する水溶液中に滴下することによりビーズ状の
ゲルが得られる。1. Bead-shaped gel can be obtained by dropping it into an aqueous solution containing calcium ions from a nozzle such as a syringe or pipette.
２、 カルシウムイオンを含有する水溶液中で注射器、
ピペット等のノズルから連続的に吐出させることにより
繊維状のゲルが得られる。2. Syringe in an aqueous solution containing calcium ions,
A fibrous gel can be obtained by continuously discharging it from a nozzle such as a pipette.
３、 平板上にキャストするか濾紙もしくはガーゼなど
に含浸させた後、水溶液中のカルシウムイオンと接触さ
せることにより膜状のゲルを得ることができる。3. After casting on a flat plate or impregnating filter paper or gauze, a membrane-like gel can be obtained by contacting with calcium ions in an aqueous solution.
ここに得られた固定化ゲルはゲル内に安定化された多数
の空ゲキを有しており、動物細胞等をかなり長期間にわ
たって自由に増殖させることができ、各種生理活性物質
等を大量生産することができるものである。The immobilized gel obtained here has a large number of cells stabilized within the gel, allowing animal cells to grow freely over a fairly long period of time, and making it possible to mass-produce various physiologically active substances. It is something that can be done.
次に本発明の実施例を示す。Next, examples of the present invention will be shown.
実施例１
重ソウ０．０５％を含むアルギン酸ナトリウム（Ｍ／Ｇ
比１．０．１％粘度１００ｃｐ／２５℃）に、市販のマ
ウスミエローマ細胞（大日本製薬に、に製）にＢリンパ
球を常法により融合したハイブリドーマ培養懸濁液を１
、ＯＸ １０’ケ／ｇゲルになるよう添加、混合し、最
終的にハイブリドーマを温潤させた１％アルギン酸ナト
リウム溶液を得た。Example 1 Sodium alginate (M/G) containing 0.05% sodium chloride
A hybridoma culture suspension obtained by fusion of B lymphocytes to commercially available mouse myeloma cells (manufactured by Dainippon Pharmaceutical Co., Ltd.) using a conventional method was added to
, OX was added and mixed to give 10'kg of gel, and finally a 1% sodium alginate solution was obtained which warmed the hybridoma.
この１％アルギン酸ナトリウム溶液を注射器につめ、氷
水中の０．１Ｍ塩化カルシウム溶液に滴下し６０分放置
し、空ゲキが多数できた粒状化ゲルを得た。This 1% sodium alginate solution was filled into a syringe, and dropped into a 0.1M calcium chloride solution in ice water and left for 60 minutes to obtain a granulated gel with many hollow particles.
得られたゲルを、下記の組成の無血清ＲＤＦ培地で３日
間３７℃で静置培養し、グルコースの消費量をみたとこ
ろ、２０日間定量的にグルコースを消費しているのが認
められた。When the obtained gel was statically cultured at 37° C. for 3 days in a serum-free RDF medium having the following composition and the amount of glucose consumed was observed, it was observed that glucose was consumed quantitatively for 20 days.
ＲＤＦ培地組成
ＲＰＭＩ １６４０ ５．４６ｇＤＭＥ
２．６２ｇＦ１２
２．７８ｇ
ＨＥＰＥＳ ０．３５７ｇＮａＨＣＯ
３１、０５ｇ
Ｐｅｎ１ｃｉｌｌｉｎ １０ｓｕｎ１０５
ｕｎｉｔＳｔｒｅｐｔｏ ０．１ｇ ｕｎｉ
ｔＩｎｓｕｌｉｎ ｌ０ＨＴｒａｎｓｆ
ｅｒｒｉｎ ２０ＢＭｏｎｏｅｔｈａｎｏｌ
ａｍｉｎｅ ２０ μｍｏｌＳｏｄｉｕｍ ５ｅ
ｌｅｎｉｔｅ １ ｎｍｏｌＷａｔｅｒ
１１ｉｔｅｒ実施例２
粒径８４μｍを有するデンプン粒０．１％ Ｖ／Ｖ及び
ヘキサメタリン酸ナトリウム０．０１％を含む１．０％
アルギン酸ナトリウム（Ｍ／Ｇ比０．２．１％粘度５０
ｃｐ／ ２５℃）溶液５０ｄを０．１Ｍ塩化カルシウム
溶液に滴下し、ビーズ状のゲルを得た。RDF medium composition RPMI 1640 5.46gDME
2.62gF12
2.78g HEPES 0.357g NaHCO
31, 05g Pen1cillin 10sun105
unitStrepto 0.1g uni
tInsulin l0HTTransf
errin 20B Monoethanol
amine 20 μmolSodium 5e
lenite 1 nmolWater
11iter Example 2 Starch granules with particle size 84 μm 0.1% V/V and 1.0% containing 0.01% sodium hexametaphosphate
Sodium alginate (M/G ratio 0.2.1% viscosity 50
cp/25°C) solution was dropped into a 0.1M calcium chloride solution to obtain a bead-shaped gel.
得られたゲル内には、デンプン粒から生成した針状の空
ゲキが中心部に向って多数みられた。In the resulting gel, many needle-like cavities generated from starch granules were observed toward the center.
実施例３
重ソウＯ，ＯＳ％を含むアルギン酸ナトリウム（Ｍ／Ｇ
比０．２．１％粘度５０ｃｐ／２５℃）溶液１００ｍ１
２に市販のマウスミエローマ細胞（大日本製薬に、に製
）にＢリンパ球を常法により融合したハイブリドーマ培
養懸濁液を１．ＯＸ １０＠ケ／ｇゲルになるよう添加
し、更に平均粒径８０μｍを有するガラスビーズを０．
１％Ｖ／Ｖになるよう添加、両者を混合し、最終的にハ
イブリドーマ及びガラスビーズを懸濁させた１％アルギ
ン酸ナトリウム溶液を得た。 この１％アルギン酸ナト
リウム溶液を注射器につめ、氷水中の０．１Ｍ塩化カル
シウム溶液に滴下し、６０分放置し、大きな空ゲキを多
数有する粒状化ゲルを得た。Example 3 Sodium alginate (M/G
0.2.1% viscosity 50cp/25℃) solution 100ml
2. A hybridoma culture suspension in which B lymphocytes were fused to commercially available mouse myeloma cells (manufactured by Dainippon Pharmaceutical Co., Ltd.) using a conventional method was added to 1. OX was added to give 10 ml/g gel, and glass beads having an average particle size of 80 μm were added to 0.0 μm.
The mixture was added to give a 1% V/V and the two were mixed to finally obtain a 1% sodium alginate solution in which hybridomas and glass beads were suspended. This 1% sodium alginate solution was filled into a syringe, added dropwise to a 0.1M calcium chloride solution in ice water, and left to stand for 60 minutes to obtain a granulated gel having many large cavities.
得られたゲルを前述のＲＤＦ培地で３日間３７℃で静地
培養し、グルコースの消費量をみたところ、２０日間定
量的にグルコースを消費しているのが認められた。When the obtained gel was statically cultured at 37° C. for 3 days in the above-mentioned RDF medium and the amount of glucose consumed was observed, it was observed that glucose was consumed quantitatively for 20 days.
代理人 弁理士 戸 １）親 男Agent Patent Attorney 1) Parent Male
Claims (1)
１５００ｃｐ（２５℃における）であるアルギン酸ナト
リウムの０．１〜２％の溶液に、キレート剤及び動物細
胞、細胞内小器官、植物細胞、植物の不定胚、デンプン
、ガラスビーズ、活性炭、シリカゲル、イオン交換樹脂
などの微粒子の１種以上を添加したものをカルシウムイ
オンで適宜形状に成型することを特徴とする空ゲキを有
するゲルの製造方法。(1) M/G ratio is 0.2 to 1.5, 1% solution is 10 to
Chelating agents and animal cells, intracellular organelles, plant cells, plant somatic embryos, starch, glass beads, activated carbon, silica gel, ions, in a 0.1-2% solution of sodium alginate that is 1500 cp (at 25 °C) A method for producing a gel having air bubbles, which comprises molding a gel containing one or more types of fine particles such as an exchange resin into an appropriate shape using calcium ions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5383488A JPH01229048A (en) | 1988-03-09 | 1988-03-09 | Production of gel |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5383488A JPH01229048A (en) | 1988-03-09 | 1988-03-09 | Production of gel |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01229048A true JPH01229048A (en) | 1989-09-12 |
| JPH0571620B2 JPH0571620B2 (en) | 1993-10-07 |
Family
ID=12953819
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5383488A Granted JPH01229048A (en) | 1988-03-09 | 1988-03-09 | Production of gel |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01229048A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996011230A1 (en) * | 1994-10-05 | 1996-04-18 | Monsanto Company | Alginate gels |
| JP2007505197A (en) * | 2003-05-23 | 2007-03-08 | ドクトル ズベラック スキン アンド ヘルス ケア アクチェンゲゼルシャフト | Process for the preparation of alginate-containing porous articles |
| JP2007106965A (en) * | 2005-10-17 | 2007-04-26 | Asahi Kasei Corp | Gel particle |
| CN114522157A (en) * | 2022-02-23 | 2022-05-24 | 重庆大学 | Application of calcium ion chelating agent in preparation of preparation for improving phagocytic capacity of vascular endothelial cells |
-
1988
- 1988-03-09 JP JP5383488A patent/JPH01229048A/en active Granted
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996011230A1 (en) * | 1994-10-05 | 1996-04-18 | Monsanto Company | Alginate gels |
| JP2007505197A (en) * | 2003-05-23 | 2007-03-08 | ドクトル ズベラック スキン アンド ヘルス ケア アクチェンゲゼルシャフト | Process for the preparation of alginate-containing porous articles |
| JP2007106965A (en) * | 2005-10-17 | 2007-04-26 | Asahi Kasei Corp | Gel particle |
| JP4623731B2 (en) * | 2005-10-17 | 2011-02-02 | 旭化成株式会社 | Gel particles |
| CN114522157A (en) * | 2022-02-23 | 2022-05-24 | 重庆大学 | Application of calcium ion chelating agent in preparation of preparation for improving phagocytic capacity of vascular endothelial cells |
| CN114522157B (en) * | 2022-02-23 | 2023-08-15 | 重庆大学 | Application of calcium ion chelating agent in preparation of preparation for improving phagocytic capacity of vascular endothelial cells |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0571620B2 (en) | 1993-10-07 |
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