KR910007847B1 - New sponge-like microporous - Google Patents

New sponge-like microporous Download PDF

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KR910007847B1
KR910007847B1 KR1019890008030A KR890008030A KR910007847B1 KR 910007847 B1 KR910007847 B1 KR 910007847B1 KR 1019890008030 A KR1019890008030 A KR 1019890008030A KR 890008030 A KR890008030 A KR 890008030A KR 910007847 B1 KR910007847 B1 KR 910007847B1
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carrier
particulate carrier
gelatin
porous
diameter
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KR910001031A (en
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김정희
최준호
임현수
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한국과학기술원
이상수
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Abstract

A process for preparing sponge-like macroporous gelatin microcarrier (I) comprises: (a) mixing gelatin soln. with forming agent, e.g., NaHCO3 or CaCO3 (The particle size is 20-30 μm or less), and adding the mixed soln of chloroform (II) and toluene (III) contg. 1-4% surfactant. (mixing ration of (II):(III) is 3:7); (b) agitating the mixt. at 300-1000rpm to form emulsion; (c) filtering it with 100 μm sieve and washing with D.W.; (d) cross-linking with glutaraldehyde, and drying to obtain the final product. The diameter of (I) is 150-500 μm and pore size of (I) is 20-50 μm.

Description

스폰지 구조를 갖는 새로운 다공성 젤라틴 미립 담체 및 그 제조방법New porous gelatin particulate carrier with sponge structure and method for producing same

제1도는 본 발명에 의하여 얻은 젤라틴을 주재료로 한 신규의 다공성 미립 담체의 주사 현미경 사진(배율 500배)이다.1 is a scanning micrograph (magnification of 500 times) of a novel porous particulate carrier based on gelatin obtained according to the present invention.

제2도는 제1도에 도시된 미립 담체중의 구멍 조직의 주사 현미경 사진(배율 2000배)이다.2 is a scanning micrograph (magnification 2000 times) of pore tissue in the particulate carrier shown in FIG.

본 발명은 세포 배양 등에 사용되는 스폰지 구조를 갖는 새로운 다공성 미립 담체(spong-like macroporous microcarrier) 및 그 제조 방법에 관한 것이다.The present invention relates to a novel sponge-like macroporous microcarrier having a sponge structure for use in cell culture and the like and a method of manufacturing the same.

기존의 미립 담체(solid microcarrier)는 주로 부착성 동물 세포의 배양에 사용되어 왔으며 대부분이 표면 부착성 미립 담체로서 담체 자체 또는 담체 표면의 전하량이나 물질을 조절·변화시킴으로써 세포의 성장에 필요한 표면(substrate)의 역할을 수행하여 왔다. 이 표면 부착성 미립 담체를 이용한 세포 배양시에는, 초기의 세포 접종량이 많이 요구될 뿐만 아니라, 담체의 표면적이 한정되어 있어 반응기 내의 세포의 농도가 낮은편이다 (3×106cells/ml). 더우기, 교반식 세포 반응기 내에서 세포를 배양할 때 담체간의 충돌 및 담체 주변에 생성되는 와류 또는 전단 응력 등의 수력학적인 영향(hydrodynamic effect) 때문에, 어느정도 이상의 교반에서는 세포가 다치게 되어 죽어버리는 현상이 발생하는 단점이 있다.Conventional solid microcarriers have been mainly used for culturing adherent animal cells, and most of them are surface adherent particulate carriers, which are required for the growth of cells by controlling or changing the amount or material of the carrier itself or the surface of the carrier. It has played the role of). In the cell culture using this surface-adherent particulate carrier, not only the initial cell inoculation amount is required but also the surface area of the carrier is limited, so that the concentration of cells in the reactor is low (3 × 10 6 cells / ml). Moreover, due to hydrodynamic effects such as collisions between carriers and vortex or shear stresses generated around the carriers when the cells are cultured in a stirred cell reactor, the cells may be injured and die at a certain agitation. There is a disadvantage that occurs.

이상과 같은 기존의 표면 부착성 미립 담체의 단점을 보완하기 위하여 세계 각국에서는 세포가 담체의 내부에서 증식할 수 있는 새로운 형태의 스폰지 구조를 갖는 다공성의 미립 담체의 개발에 노력을 경주하고 있다. 1986년에 미국의 베락스사(Verax Corporaton)에서 고가의 콜라겐(collagen)을 이용하여 다공성 미립담체의 개발에 성공하여 현재 산업체에서 사용 중에 있다. 그러나, 재질인 콜라겐은 세포에 의해 미량 분비되는 고가의 물질이기 때문에, 최근 스웨덴이나 미국 등을 포함한 선진 각국에서는 좀더 저렵한 대체 물질을 찾는 데 많은 연구가 경주되고 있다. 1986년 스웨덴의 모스바흐(K.Mosbach)등이 동물의 연골 또는 근육에 다량 존재하는 젤라틴을 이용하여 다공성의 미립 담체를 개발했다는 보고가 있다(K.Mosbach, 1986, Biotechnology, 4, 989, 990). 그러나, 전자 현미경 사진과 세포 배양 결과를 토대로 평가해 본 결과, 이 담체의 표면에 어느 정도의 굴곡이 존재하여 다공성처럼 보이기는 하지만, 실제로 세포가 담체의 내부에까지 들어가 증식할 수 있는 스폰지와 같은 다공성 담체의 개발에 성공한 것은 아니었다.In order to supplement the above disadvantages of the conventional surface-adherent particulate carriers, efforts are being made in the development of porous particulate carriers having a new type of sponge structure in which cells can proliferate inside the carrier. In 1986, Verax Corporaton of the United States succeeded in the development of porous particulate carrier using expensive collagen (collagen), currently in use in industry. However, since collagen, a material, is an expensive substance that is secreted by the cells in a small amount, much research has recently been conducted to find more cheap substitutes in developed countries including Sweden and the United States. In 1986, Mosbach, Sweden, and others reported that they developed porous particulate carriers using gelatin that is present in large amounts in cartilage or muscle of animals (K.Mosbach, 1986, Biotechnology, 4, 989, 990). ). However, based on electron micrographs and cell culture results, although some curvature is present on the surface of the carrier, it appears to be porous. The development of the carrier was not successful.

본 발명은 이상과 같은 기존의 다공성 미립 담체의 단점이 극복된 스폰지 구조의 미립 담체 및 그 제조방법을 제공하고자 함에 그 목적이 있다.It is an object of the present invention to provide a particulate carrier having a sponge structure and a method of manufacturing the same, in which the disadvantages of the conventional porous particulate carrier as described above are overcome.

이러한 본 발명의 목적은 젤라틴 용액을 클로로포름/톨루엔(3:7)의 혼합 유기 용매에 에멀젼시키면서 적당한 발포제를 가하여 스폰지와 같은 구멍 조직으로 이루어진 둥근 모양의 다공성 미립담체를 얻은 다음 글루타르알데히드로 가교 결합(cross-linking)시킴으로써 안정한 구조를 갖는 다공성 미립 담체를 얻음으로써 달성된다. 본 발명에 사용되는 젤라틴 용액은 7-25%인 것이 바람직하다. 또, 발포제로서는 NaHCO3나 CaCO3등의 공지의 CO2발생 발포제를 사용하는 것이 좋다. 글루타르 알데히드 용액은 농도 3-7%의 것이 사용된다. 클로로포름/톨루엔 혼합 용매에는 Tween 80, Arlacel 83, Span 85(이상”상표명)등의 계면 활성제를 함유시키는 것이 균질한 발포체를 얻는데 좋다. 본 발명의 다공성 미립 담체는 용액 상태나 건조상태의 어느 경우에도 안정할 뿐만 아니라, 고온 살균(121。C에서 15분)에서도 매우 안정하다.The object of the present invention is to add a suitable blowing agent while emulsifying the gelatin solution to a mixed organic solvent of chloroform / toluene (3: 7) to obtain a round porous microcarrier composed of a sponge-like pore structure, and then glutaraldehyde crosslinking. by cross-linking to obtain a porous particulate carrier having a stable structure. The gelatin solution used in the present invention is preferably 7-25%. As the blowing agent, it is preferable to use a known CO 2 generating blowing agent such as NaHCO 3 or CaCO 3 . Glutar aldehyde solution is used with a concentration of 3-7%. Incorporating surfactants such as Tween 80, Arlacel 83, and Span 85 (trade name) in the chloroform / toluene mixed solvent is preferable to obtain a homogeneous foam. The porous particulate carrier of the present invention is not only stable in either the solution state or in the dry state, but also very stable at high temperature sterilization (15 minutes at 121 ° C.).

본 발명에서는 발포제의 크기와 성질을 조절함으로써 다공성 미립 담체의 구멍 조직의 직경을 20-50μm로 조절할 수 있을 뿐만 아니라, 가교 결합시키는 반응의 정도에 따라 기계적 강도도 조절할 수 있다. 특히, 기존의 미립 담체에 비해 월등히 넓은 표면적과 담체 내부가 스폰지와 같은 구멍 조직으로 되어 있어, 실제의 조직과 유사한 환경이 제공되고, 또 전단 응력 등과 같은 배양 환경으로부터 세포들이 보호되기 때문에, 5g/ℓ의 다공성 미립 담체를 사용하였을 때 기존의 미립 담체에 비해 4-5배의 높은 세포 농도를 얻을 수 있다. 또한, 본 발명에 따른 새로운 형태의 스폰지 구조를 갖는 다공성 미립담체를 부착성 동물 세포 뿐만 아니라, 하이브리도마 세포와 같은 부유성 동물 세포의 배양에도 사용될 수 있다.In the present invention, by adjusting the size and properties of the blowing agent, not only the diameter of the pore tissue of the porous particulate carrier can be adjusted to 20-50 μm, but also the mechanical strength according to the degree of the crosslinking reaction. In particular, 5 g / When using a porous particulate carrier of 4L it can be obtained a higher cell concentration of 4-5 times than the conventional particulate carrier. In addition, the porous particulate carrier having a novel type of sponge structure according to the present invention can be used for the cultivation of not only adherent animal cells but also floating animal cells such as hybridoma cells.

본 발명에서 제조되는 스폰지 구조의 다공성 젤라틴 미립 담체의 일반적 특성은 다음과 같다.General characteristics of the porous gelatin particulate carrier of the sponge structure prepared in the present invention are as follows.

(ㄱ) 모양 : 구형 (球形)(A) Shape: spherical

(ㄴ) 담체의 직경 : 150-500μm(B) Diameter of carrier: 150-500μm

(ㄷ) 구멍 조직의 직경(pore size) : 20-50μm(C) Pore size: 20-50μm

(ㄹ) 비중 : 1.2-1.6(D) Specific gravity: 1.2-1.6

(ㅁ) 표면적 : 7-10m2/g(ㅁ) Surface area: 7-10m 2 / g

(ㅂ) 세포에 대해 무독성이며, 광학적으로 투명(Iii) non-toxic and optically transparent to cells

(ㅅ) 콜라게나제(collagenase), 디스파제(dispase) 등의 효소에 의해 분해 가능(G) Degradable by enzymes such as collagenase and dispase

(ㅇ) 분말 또는 용액 상태로 살균 기능(ㅇ) Sterilization function in powder or solution state

본 발명을 실시예에 따라 상술하면 다음과 같다.Hereinafter, the present invention will be described in detail as follows.

[실시예1]Example 1

10%(w/v)의 젤라틴 용액에 입도가20μm 미만인 NaHCO3(또는 CaCO3)를 10-20%첨가하여 혼합한 다음, Tween 80 1-2%가 함유된 클로로포름/톨루엔(3 : 7)의 혼합 용매 100ml에 첨가하여 700-800rpm으로 교반하여 에멀젼을 형성하였다. 형성된 에멀젼을 100μm의 체를 이용하여 여과한 다음 증류수로 세척하였다. 회수된 미립담체 내부에 포집된 발포제를 발포시킨 다음, 최종 농도 2-4%의 글루타르알데히드로 30분간 가교 결합 반응시켜서 다공성의 미립 담체를 얻고, 이어서 최종 상태로 동결 건조를 거쳐 건조된 분말 상태의 미립 담체를 얻었다. 이때, 구멍 조직의 직경은 20-30μm 이었으며, 담체의 직경은 150-250μm이었다.10% (w / v) gelatin solution was mixed with 10-20% NaHCO 3 (or CaCO 3 ) with a particle size of less than 20 μm, followed by chloroform / toluene (3: 7) containing 1-2% of Tween 80. Was added to 100 ml of a mixed solvent and stirred at 700-800 rpm to form an emulsion. The resulting emulsion was filtered using a 100 μm sieve and then washed with distilled water. The foamed agent collected in the recovered particulate carrier was foamed, and then crosslinked for 30 minutes in glutaraldehyde at a final concentration of 2 to 4% to obtain a porous particulate carrier, followed by lyophilization to a final state and dried in powder form. A particulate carrier of was obtained. At this time, the diameter of the pore tissue was 20-30μm, the diameter of the carrier was 150-250μm.

[실시예2]Example 2

교반 속도를 300-400rpm으로 하는 것을 제외하고는 실시예1의 방법을 반복하였다. 담체의 평균 직경이 500μm인 미립 담체가 생성되었다. 이 때의 구멍 조직의 직경은 20-40μm이었다.The method of Example 1 was repeated except that the stirring speed was 300-400 rpm. A particulate carrier with a mean diameter of 500 μm was produced. The diameter of the hole structure at this time was 20-40 micrometers.

[실시예3]Example 3

교반 속도를 1000rpm으로 바꾸어 미립 담체의 직경이 120-150μm인 다공성 미립담체를 제조하였다.A porous particulate carrier having a diameter of 120-150 μm was prepared by changing the stirring speed to 1000 rpm.

[실시예4]Example 4

실시예 1에서 입도가 평균 30μm인 발포제(NaHCO3또는 CaCO3)를 사용하는 것을 제외하고는, 실시예 1의 방법을 수행하여 구멍 조직의 직경이 40-50μm인 다공성의 미립 담체를 얻었다.Except for using a blowing agent (NaHCO 3 or CaCO 3 ) having an average particle size of 30 μm in Example 1, the method of Example 1 was carried out to obtain a porous particulate carrier having a diameter of 40-50 μm of pore tissue.

[실시예5]Example 5

실시예 1의 방법을 이용하되, 젤라틴 용액의 농도를 7-25%로 하여 동일한 다공성의 미립 담체를 얻었다.Using the method of Example 1, the granular carrier of the same porosity was obtained by setting the concentration of gelatin solution to 7-25%.

[실시예6]Example 6

실시예1에서의 Tween 80 1-2% 대신에 Arlacel 83(또는 Span 85) 1-4%를 사용하여 시실시예 1과 동일한 다공성의 미립 담체를 얻었다.1-4% of Arlacel 83 (or Span 85) instead of 1-2% of Tween 80 in Example 1 was used to obtain the same porous particulate carrier as in Example 1.

[실시예7]Example 7

실시예 1에서의 동결 건조 방법 대신 스프레이 건조기(spray dryer)를 이용하여 동일한 형태의 다공성 미립 담체를 제조하였다.Instead of the lyophilization method in Example 1, a spray dryer was used to prepare porous particulate carriers of the same type.

Claims (6)

다음 특성을 갖는 스폰지 구조를 갖는 젤라틴 다공성 미립 담체.A gelatin porous particulate carrier having a sponge structure having the following characteristics. (ㄱ) 모양 : 구형(球形)(A) Shape: spherical (ㄴ) 담체의 직경 : 150-500μm(B) Diameter of carrier: 150-500μm (ㄷ) 구멍 조직의 직경(pore size) : 20-50μm(C) Pore size: 20-50μm (ㄹ) 비중 : 1.2-1.6(D) Specific gravity: 1.2-1.6 (ㅁ) 표면적 : 7-10m2/g(ㅁ) Surface area: 7-10m 2 / g (ㅂ) 광학적으로 투명(Iii) optically transparent (ㅅ) 콜라게나제(collagenase), 디스파제(dispase) 등의 효소에 의해 분해 가능(G) Degradable by enzymes such as collagenase and dispase (ㅇ) 분말 또는 용액 상태로 살균 가능(O) Possible to sterilize in powder or solution state 발포제 존재하에 젤라틴 용액을 클로로포름/톨루엔(3 : 7)의 혼합 용매 중에서 교반하여 에멀젼화시킨 다음, 글루타르알데히드를 가하여 가교 결합 반응시키는 것을 특징으로 하는 다음 특성의 스폰지 구조를 갖는 다공성 젤라틴 미립 담체의 제조 방법.The gelatin solution in the presence of a blowing agent is emulsified by stirring in a mixed solvent of chloroform / toluene (3: 7), and then subjected to crosslinking reaction by adding glutaraldehyde to obtain a porous gelatin particulate carrier having a sponge structure having the following characteristics. Manufacturing method. (ㄱ) 모양 : 구형(球形)(A) Shape: spherical (ㄴ) 담체의 직경 : 150-500μm(B) Diameter of carrier: 150-500μm (ㄷ) 구멍 조직의 직경(pore size) : 20-50μm(C) Pore size: 20-50μm (ㄹ) 비중 : 1.2-1.6(D) Specific gravity: 1.2-1.6 (ㅁ) 표면적 : 7-10m2/g(ㅁ) Surface area: 7-10m 2 / g (ㅂ) 광학적으로 투명(Iii) optically transparent (ㅅ) 콜라게나제(collagenase), 디스파제(dispase) 등의 효소에 의해 분해 가능(G) Degradable by enzymes such as collagenase and dispase (ㅇ) 분말 또는 요액 상태로 살균 가능(ㅇ) Sterilization in powder or urine state 제2항에 있어서, 발포제가 입도 20-30μm의 NaHCO3또는 CaCO3인 것인 방법.The method of claim 2 wherein the blowing agent is NaHCO 3 or CaCO 3 with a particle size of 20-30 μm. 제2항에 있어서, 교반 속도가 300-1000rpm으로 조절됨으로써 다공성 미립 담체의 크기가 150-500μm으로 조절되는 것인 방법.The method of claim 2, wherein the size of the porous particulate carrier is controlled to 150-500 μm by controlling the stirring speed to 300-1000 rpm. 제2항에 있어서, 혼합 용매에 계면 활성제가 1-4% 함유되는 것인 방법.The method of claim 2 wherein the mixed solvent contains 1-4% of a surfactant. 제2항에 있어서, 미립 담체 제품이 동결 전조 또는 스프레이 건조되는 것인 방법.The method of claim 2, wherein the particulate carrier product is freeze precursor or spray dried.
KR1019890008030A 1989-06-10 1989-06-10 New sponge-like microporous KR910007847B1 (en)

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Cited By (7)

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US9005609B2 (en) 2003-08-07 2015-04-14 Ethicon, Inc. Hemostatic compositions containing sterile thrombin
US9821025B2 (en) 2011-10-11 2017-11-21 Baxter International Inc. Hemostatic compositions
US9833541B2 (en) 2011-10-27 2017-12-05 Baxter International Inc. Hemostatic compositions
US9999703B2 (en) 2012-06-12 2018-06-19 Ferrosan Medical Devices A/S Dry haemostatic composition
US10111980B2 (en) 2013-12-11 2018-10-30 Ferrosan Medical Devices A/S Dry composition comprising an extrusion enhancer
US10245348B2 (en) 2010-06-01 2019-04-02 Baxter International Inc. Process for making dry and stable hemostatic compositions
US10322170B2 (en) 2011-10-11 2019-06-18 Baxter International Inc. Hemostatic compositions

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9005609B2 (en) 2003-08-07 2015-04-14 Ethicon, Inc. Hemostatic compositions containing sterile thrombin
US10245348B2 (en) 2010-06-01 2019-04-02 Baxter International Inc. Process for making dry and stable hemostatic compositions
US9821025B2 (en) 2011-10-11 2017-11-21 Baxter International Inc. Hemostatic compositions
US10322170B2 (en) 2011-10-11 2019-06-18 Baxter International Inc. Hemostatic compositions
US9833541B2 (en) 2011-10-27 2017-12-05 Baxter International Inc. Hemostatic compositions
US9999703B2 (en) 2012-06-12 2018-06-19 Ferrosan Medical Devices A/S Dry haemostatic composition
US10111980B2 (en) 2013-12-11 2018-10-30 Ferrosan Medical Devices A/S Dry composition comprising an extrusion enhancer

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