KR910007847B1 - New sponge-like microporous - Google Patents

New sponge-like microporous Download PDF

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KR910007847B1
KR910007847B1 KR890008030A KR890008030A KR910007847B1 KR 910007847 B1 KR910007847 B1 KR 910007847B1 KR 890008030 A KR890008030 A KR 890008030A KR 890008030 A KR890008030 A KR 890008030A KR 910007847 B1 KR910007847 B1 KR 910007847B1
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carrier
diameter
particulate carrier
method
dispase
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KR910001031A (en )
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김정희
최준호
임현수
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이상수
한국과학기술원
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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 제조방법 New porous particulate carrier and a method of manufacturing gelatin having a sponge structure

제1도는 본 발명에 의하여 얻은 젤라틴을 주재료로 한 신규의 다공성 미립 담체의 주사 현미경 사진(배율 500배)이다. The first turn is a scanning micrograph (magnification 500X) of the porous particulate carrier of a main material novel gelatin obtained by the present invention.

제2도는 제1도에 도시된 미립 담체중의 구멍 조직의 주사 현미경 사진(배율 2000배)이다. 2 is a scanning microscopic photograph (magnification 2000 times) of the hole in the tissue of the particulate carrier shown in FIG. 1 turn.

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 porous particulate carrier (spong-like macroporous microcarrier) and a method of manufacturing the same having a sponge structure used in cell culture.

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. Conventional particulate carrier (solid microcarrier) has been mainly used in the adherent culture of an animal cell surface mostly by surface adhesion control, changing the amount of charge or the material of the support itself or the carrier surface as a particulate carrier required for the growth of cells (substrate ) it has to do the role. 이 표면 부착성 미립 담체를 이용한 세포 배양시에는, 초기의 세포 접종량이 많이 요구될 뿐만 아니라, 담체의 표면적이 한정되어 있어 반응기 내의 세포의 농도가 낮은편이다 (3×10 6 cells/ml). The surface adhesion during cell culture using a particulate carrier, as well as demands a lot of the beginning of the cell inoculum size, the surface area of the carrier is limited, there is a cell with low concentration side of the reactor (3 × 10 6 cells / 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. Moreover, since the stirred cell reactor within hydrodynamic effects (hydrodynamic effect), such as eddy current or the shear stress generated cells around collision and carrier between the carrier when cultured in, the symptoms in some degree stirred over discard cells are injured dead there are drawbacks to occur.

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. In the world cellular and racing efforts on the development of the porous particulate carrier has a sponge structure of a new type that can be propagated in the interior of the carrier in order to compensate for the shortcomings of the existing surface adhesion particulate carrier as described above. 1986년에 미국의 베락스사(Verax Corporaton)에서 고가의 콜라겐(collagen)을 이용하여 다공성 미립담체의 개발에 성공하여 현재 산업체에서 사용 중에 있다. In 1986. In Ephesus Lux Corporation (Verax Corporaton) in the United States use of expensive collagen (collagen) it succeeded in developing a porous particulate carriers are being currently used in the industry. 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. However, the collagen material is expensive because of the trace substances secreted by the cells, recent advanced countries, including Sweden and the United States has been a lot of research 10000000000000000 weeks to find a better alternative substances China. 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). Etc. 1986 Moss Bach (K.Mosbach) Sweden has reported that the development of a porous particulate carrier using a large amount of gelatin present in the cartilage or muscle of animals (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. However, the results of this year evaluated based on an electron microscope photograph with cell culture results, and the surface of the carrier, there is a certain degree of curvature of looks like a porous, but actually porous as a sponge which cells can grow into the far interior of the carrier the successful development of the carrier was not.

본 발명은 이상과 같은 기존의 다공성 미립 담체의 단점이 극복된 스폰지 구조의 미립 담체 및 그 제조방법을 제공하고자 함에 그 목적이 있다. The present invention has as its object to provide a conventional porous fine particulate carrier of the sponge structure overcomes drawbacks of the support and a manufacturing method as described above.

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. Cross-linked to the next glutaraldehyde while the emulsion to a mixed organic solvent by adding a suitable blowing agent obtained a porous particulate carrier of round, made of a hole in tissue, such as sponges: The object of the present invention is a gelatine solution of chloroform / toluene (73) by (cross-linking) is achieved by obtaining a porous particulate carrier having a stable structure. 본 발명에 사용되는 젤라틴 용액은 7-25%인 것이 바람직하다. Gelatin solution for use in the invention is preferably from 7-25%. 또, 발포제로서는 NaHCO 3 나 CaCO 3 등의 공지의 CO 2 발생 발포제를 사용하는 것이 좋다. Further, as the blowing agent it is better to use a CO 2 blowing agent generating a known, such as NaHCO 3 or CaCO 3. 글루타르 알데히드 용액은 농도 3-7%의 것이 사용된다. Glutaraldehyde solution is used to a concentration of 3-7%. 클로로포름/톨루엔 혼합 용매에는 Tween 80, Arlacel 83, Span 85(이상”상표명)등의 계면 활성제를 함유시키는 것이 균질한 발포체를 얻는데 좋다. Chloroform / toluene mixed solvent, it is good to obtain a homogeneous foam comprising adding a surfactant such as Tween 80, Arlacel 83, Span 85 (or higher "Trade Mark). 본 발명의 다공성 미립 담체는 용액 상태나 건조상태의 어느 경우에도 안정할 뿐만 아니라, 고온 살균(121。C에서 15분)에서도 매우 안정하다. The porous particulate carrier of the present invention are not only stable in either case of the solution state or a dry state, it is very stable in 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. In the present invention, it may also be adjusted according to the degree of mechanical strength to the reaction not only can adjust the diameter of the fine particles of porous carrier hole tissue by controlling the size and nature of the blowing agent as 20-50μm, crosslinking. 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. In particular, since it than conventional particulate carrier significantly large surface area and carrier inside is a hole organization, such as a sponge, it is provided with a physical organization and a similar environment, and to cells are protected from the culture conditions such as shear stress, 5g / when using a porous particulate carrier of ℓ it can be obtained a high cell concentration of 4-5 fold compared to conventional particulate carrier. 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. Further, the adhesion, as well as animal cells, the porous particulate carrier has a sponge structure of the new type according to the present invention, it can be used for sub-culture of animal cells, such as oil-based hybridoma cells.

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

(ㄱ) 모양 : 구형 (球形) (A) shape: spherical (球形)

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

(ㄷ) 구멍 조직의 직경(pore size) : 20-50μm (C) the diameter of the hole organization (pore size): 20-50μm

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

(ㅁ) 표면적 : 7-10m 2 /g (E) a specific surface area: 7-10m 2 / g

(ㅂ) 세포에 대해 무독성이며, 광학적으로 투명 (F) a non-toxic for the cells, an optically transparent

(ㅅ) 콜라게나제(collagenase), 디스파제(dispase) 등의 효소에 의해 분해 가능 (Oi) can be decomposed by an enzyme, such as collagenase (collagenase), dispase (dispase)

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

본 발명을 실시예에 따라 상술하면 다음과 같다. If the above-mentioned according to the invention to the embodiment as follows.

[실시예1] Example 1

10%(w/v)의 젤라틴 용액에 입도가20μm 미만인 NaHCO 3 (또는 CaCO 3 )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. 10% (w / v) gelatin solution in a particle size less than 20μm of NaHCO 3 (or CaCO 3) 10 to 20% was added and mixed, and then, Tween 80 containing 1-2% of a chloroform / toluene (3: 7) by the addition of a mixed solvent of 100ml to form an emulsion by stirring at 700-800rpm. 형성된 에멀젼을 100μm의 체를 이용하여 여과한 다음 증류수로 세척하였다. The formed emulsion was filtered using a sieve of 100μm, and then washed with distilled water. 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. Expanding the blowing agent was trapped inside the recovered particulate carrier, and then by 30 minutes cross-linking reaction with glutaraldehyde at a final concentration of 2-4% to obtain a particulate carrier of porous, then the dried powder form through a freeze-dried to a final state of fine particles to obtain a carrier. 이때, 구멍 조직의 직경은 20-30μm 이었으며, 담체의 직경은 150-250μm이었다. At this time, the diameter of the hole was organizations 20-30μm, was the diameter of the carrier is 150-250μm.

[실시예2] Example 2

교반 속도를 300-400rpm으로 하는 것을 제외하고는 실시예1의 방법을 반복하였다. It was repeated the procedure of Example 1 except that the stirring speed 300-400rpm. 담체의 평균 직경이 500μm인 미립 담체가 생성되었다. The particulate carrier of the average diameter of the carrier 500μm was formed. 이 때의 구멍 조직의 직경은 20-40μm이었다. The diameter of the hole in the organization at that time was 20-40μm.

[실시예3] Example 3

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

[실시예4] Example 4

실시예 1에서 입도가 평균 30μm인 발포제(NaHCO 3 또는 CaCO 3 )를 사용하는 것을 제외하고는, 실시예 1의 방법을 수행하여 구멍 조직의 직경이 40-50μm인 다공성의 미립 담체를 얻었다. Example 1 of the blowing agent particle size of 30μm in average but using (NaHCO 3 or CaCO 3), and is carrying out the method of Example 1 to obtain a particulate carrier of the diameter of the hole in the porous tissue 40-50μm.

[실시예5] Example 5

실시예 1의 방법을 이용하되, 젤라틴 용액의 농도를 7-25%로 하여 동일한 다공성의 미립 담체를 얻었다. Carried out, but using the method of Example 1, The concentration of the gelatin solution to 7-25% to obtain the particulate carrier of the same porosity.

[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. Example 1 in place of Arlacel 1-2% Tween 80 at 83 (or Span 85) using 1-4% to give a particulate carrier of the same porous and Sicily example 1.

[실시예7] Example 7

실시예 1에서의 동결 건조 방법 대신 스프레이 건조기(spray dryer)를 이용하여 동일한 형태의 다공성 미립 담체를 제조하였다. Example methods lyophilized instead of 1 was prepared the same shape of the porous particulate carrier, by using a spray dryer (spray dryer).

Claims (6)

  1. 다음 특성을 갖는 스폰지 구조를 갖는 젤라틴 다공성 미립 담체. Gelatin porous particulate carrier has a sponge structure with the following characteristics:
    (ㄱ) 모양 : 구형(球形) (A) shape: spherical (球形)
    (ㄴ) 담체의 직경 : 150-500μm (B) the diameter of carrier: 150-500μm
    (ㄷ) 구멍 조직의 직경(pore size) : 20-50μm (C) the diameter of the hole organization (pore size): 20-50μm
    (ㄹ) 비중 : 1.2-1.6 (D) Specific Gravity: 1.2-1.6
    (ㅁ) 표면적 : 7-10m 2 /g (E) a specific surface area: 7-10m 2 / g
    (ㅂ) 광학적으로 투명 (F) optically transparent
    (ㅅ) 콜라게나제(collagenase), 디스파제(dispase) 등의 효소에 의해 분해 가능 (Oi) can be decomposed by an enzyme, such as collagenase (collagenase), dispase (dispase)
    (ㅇ) 분말 또는 용액 상태로 살균 가능 (O) can be sterilized in powder or solution state
  2. 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 blowing agent present chloroform / toluene (3: 7) which was stirred in a mixed solvent of emulsified, and then, a porous gelatin particulate carrier has a sponge structure of the following characteristics, comprising a step of cross-linking reaction was added to glutaraldehyde method.
    (ㄱ) 모양 : 구형(球形) (A) shape: spherical (球形)
    (ㄴ) 담체의 직경 : 150-500μm (B) the diameter of carrier: 150-500μm
    (ㄷ) 구멍 조직의 직경(pore size) : 20-50μm (C) the diameter of the hole organization (pore size): 20-50μm
    (ㄹ) 비중 : 1.2-1.6 (D) Specific Gravity: 1.2-1.6
    (ㅁ) 표면적 : 7-10m 2 /g (E) a specific surface area: 7-10m 2 / g
    (ㅂ) 광학적으로 투명 (F) optically transparent
    (ㅅ) 콜라게나제(collagenase), 디스파제(dispase) 등의 효소에 의해 분해 가능 (Oi) can be decomposed by an enzyme, such as collagenase (collagenase), dispase (dispase)
    (ㅇ) 분말 또는 요액 상태로 살균 가능 (O) it can be sterilized in a powder state or yoaek
  3. 제2항에 있어서, 발포제가 입도 20-30μm의 NaHCO 3 또는 CaCO 3 인 것인 방법. The method of claim 2 wherein the blowing agent is one of NaHCO 3 or CaCO 3 particle size of 20-30μm.
  4. 제2항에 있어서, 교반 속도가 300-1000rpm으로 조절됨으로써 다공성 미립 담체의 크기가 150-500μm으로 조절되는 것인 방법. The method of claim 2, wherein the stirring speed is adjusted to 300-1000rpm whereby the size of the porous particulate carrier which is adjusted to 150-500μm.
  5. 제2항에 있어서, 혼합 용매에 계면 활성제가 1-4% 함유되는 것인 방법. The method of claim 2, wherein the surface active agent will be contained 1-4% in a mixture.
  6. 제2항에 있어서, 미립 담체 제품이 동결 전조 또는 스프레이 건조되는 것인 방법. The method of claim 2, wherein the particulate carrier product to freeze or spray drying a precursor.
KR890008030A 1989-06-10 1989-06-10 New sponge-like microporous KR910007847B1 (en)

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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

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KR101295421B1 (en) * 2012-06-12 2013-08-09 최진선 Self-assembly fire pot for multipurpose

Cited By (4)

* 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
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

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