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CO-CULTURE DEVICE AND CO-CULTURE METHOD FOR BACTERIUM SUCH AS ANAEROBIC BACTERIUM AND EPITHELIAL CELLS

Foreign code F180009620
File No. 5450
Posted date Nov 20, 2018
Country WIPO
International application number 2017JP039206
International publication number WO 2018079793
Date of international filing Oct 30, 2017
Date of international publication May 3, 2018
Priority data
  • P2016-211626 (Oct 28, 2016) JP
Title CO-CULTURE DEVICE AND CO-CULTURE METHOD FOR BACTERIUM SUCH AS ANAEROBIC BACTERIUM AND EPITHELIAL CELLS
Abstract To provide a means for co-culturing a bacterium such as an anaerobic bacterium and epithelial cells, disclosed is a culture system for co-culturing a first cell group consisting of one or more kinds of cells and a cell layer or tissue formed of a second cell group consisting of one or more kinds of cells different from the former cells, said culture system being characterized by comprising: a first culture tank for co-culturing under anaerobic conditions the first cell group consisting of one or more kinds of cells of, for example, an anaerobic bacterium and the cell layer or tissue formed of the second cell group consisting of one or more kinds of cells such as epithelial cells; a second culture tank for pooling a liquid culture medium under aerobic conditions; one or more matter exchange structures that are disposed so as to connect the first culture tank to the second culture tank; and the aforesaid cell layer or tissue that is disposed so as to cover the surface on the first culture tank side of the matter exchange structure(s).
Outline of related art and contending technology BACKGROUND ART
Intestinal bacteria, human and animal intestinal bacteria that is always present while Gliocladiunz in a general term.The number and types of bacteria in the intestine is very large, in humans, about 1000 billion per fecal 1 g the presence of bacteria in the intestine and even more, at least about one degree and the type 100, also referred to as unidentified bacteria are many.From the inside of the intestine under anaerobic conditions, many of the anaerobic bacteria are bacteria in the intestine is considered.
Intestinal bacteria are, present in the gut absorption of a variety of materials, useful for human and animal intestinal metabolites can be discharged to, the digestion of food ingested in play an auxiliary role.In addition, the bacteria in the intestine, bowel extends normally in a stable configuration the intestinal flora, entering into the intestines of the human and animal pathogens to prevent the growth of the foreign, to prevent the occurrence of a disease caused by pathogens are responsible for are considered.On the other hand, the bacteria in the intestine, as its metabolites, such as carcinogens or toxic substance, an adverse effect on human and animal survival may be the production of harmful substances.These harmful substances in the intestine of the human and animal acts directly on, the absorption from the intestine as well as systemic blood flow delivered through a variety of action and cause a disorder or disease, human and animal health is impaired.
In this way, the number and types Gliocladiunz in the gut bacteria in the intestine of the various, intestinal bacteria are intestinal flora is formed, while maintaining a state of healthy human and animal remains viable and is essential for the presence of, and even the cause of a disease, in human and animal, the quality of the health of the left and right is considered to be very important are present.
On the other hand, the number and types of bacteria in the intestine, as well as the specific structure of the intestinal flora, each individual, different for each individual, in addition, the same individual, the individual, diet and the environment such as external factors such as the condition of the living environment by the change of the internal factor of, the type of bacteria in the intestine or a variation in the ratio are needed.Therefore, the intestinal flora specific arrangement of the effect on human and animal health conditions, and continue with daily changes are considered.
In recent years, such the presence of bacteria in the intestine and the intestinal flora human and animal health conditions associated with the large and the effect of the importance of attention.Then, the intestinal flora and intestinal bacteria would have an adverse effect on health and health factors contributing to elucidate the factors, human and animal health of the intestinal bacteria and, in the treatment and prevention of various diseases and the establishment of a method a very forceful momentum demand.Therefore, in order to elucidate the picture of the intestinal flora, enteric bacteria in the intestine of the human and animal from the collected and isolated culture, attempts have been made to identify the type and characteristics, can be cultured under aerobic conditions for bacteria in the intestine of the part, the analysis has been, in the presence of intestinal epithelial cells is performed by the characteristics of the confirmation test.However, intestinal bacteria, absolute anaerobic in many cases, more than half its difficult to culture, identification of bacteria and characteristics of the check is not yet in many cases.Therefore, the type of bacteria in the intestine is applied to the variation of the ratio of the external factor, also in the relationship between the factors, is accurately known is not reached.
First of all, intestinal bacteria, substantially oxygen-free absolute anaerobic conditions in the intestine based on the fact that lived in, culture dishes were placed in an oxygen-free chamber is held in an oxygen-free conditions in the intestine is reproduced, the absolute anaerobic culturing the bacteria in the intestine in the proposed method has been used.In this method, a part of the growing in culture bacteria in the intestine is effective.However, in this method, the composition of the culture liquid in the cells need to be optimized in accordance with, on the other hand, to optimize and increase only the portion of the types of intestinal bacteria, and intestinal flora other anaerobic bacteria culture of the flame retardant is grown at the same time since it is difficult, has been required to be improved.
Therefore, the bacteria in the intestine, intestinal epithelial cells of the intestinal tract by considering the fact that a viable and in contact with each other, the bacteria in the intestine, are cultured in a culture solution is simply not, in the same manner as in the intestine, intestinal epithelial cells to coexist, intestinal epithelial cells or equal to that of the cells to create a single layer film, or, cell culture insert a cell culture system used in the gastrointestinal tract on the filter of the epithelial cells or the equivalent of cells of a single layer film having a construction of the system it is attempted.However, intestinal epithelial cells or the equivalent of cells of a single-layer film, an oxygen-free state and reproduce the environment in the intestine, a single-layer film made of the junctions between the cells in the fracture is 1, and 48 within a time cell death because the separation of the single-layer film, the intestinal epithelial cells of the intestinal tract may not be present in the culture (Patent Document 1).
Therefore, so as to mimic the natural intestinal state than the intestinal epithelial cells and bacteria in the intestine and as a co-culture, a porous film of the intestinal epithelial cells were further divided by a micro-channel, divided into upper and lower micro-channel different from the fluid source and continues to be supplied to the culture solution, the micro flow path provided on both sides of the vacuum chamber by a porous film layer of intestinal epithelial cells as stretching, intestinal bacteria culture system of intestinal epithelial cells has been proposed (Patent Document 1).
Scope of claims (In Japanese)請求の範囲 [請求項1]
 1種又は複数種の細胞から構成される第1の細胞群を、それとは異なる1種又は複数種の細胞から構成される第2の細胞群で形成された細胞層または組織と共培養するための培養システムであって、
 前記第1の細胞群を、前記第2の細胞群で形成された細胞層または組織と嫌気条件下で共培養する第1培養槽と、
 好気条件で培養液を貯留する第2培養槽と、
 前記第1培養槽と前記第2培養槽とを接続するように配される1又は複数の物質交換構造体であって、その第1培養槽側の表面を覆うように前記細胞層または組織を保持する物質交換構造体と、
 を有する、培養システム。

[請求項2]
 前記物質交換構造体を除き、前記第2培養槽の内部が閉鎖状態となるように、当該第2培養槽と前記第1培養槽との間で開放されている部分を閉じた状態とし、前記第1培養槽と前記第2培養槽との接続部にガス非透過性の封止部材を備える、請求項1に記載の培養システム。

[請求項3]
 前記第2培養槽が、当該第2培養槽において外部に開放されている部分を密封するガス透過性の保湿部材を備え、当該保湿部材で密封された部分の他に外部に開放されてない、請求項1または2に記載の培養システム。

[請求項4]
 請求項1から3までのいずれか1項に記載の培養システムであって、
 前記第1培養槽が、
  その内部に複数の部分構造と、
  前記物質交換構造体とは別の物質交換構造体と
 を備え、
 当該部分構造の各々が、培養槽として機能し、
 当該部分構造の各々が、前記別の物質交換構造体によって相互に接続され、
 当該複数の部分構造のそれぞれが、前記第2培養槽に接続している、培養システム。

[請求項5]
 請求項1から4までのいずれか1項に記載の培養システムであって、
 嫌気チェンバー内の環境に配置して使用される、培養システム。

[請求項6]
 第1培養槽と、第2培養槽とを有する、細菌を、上皮細胞から形成される細胞層との共培養を行う培養システムであって、
 前記第1培養槽は、その底面に設けられた1又は複数の物質交換構造体と、前記細胞層がその物質交換構造体の頂面を覆うようにして配されたものであり、
 前記第2培養槽は、好気条件の培養液を貯留する槽であって、前記第1の培養槽がその槽内に挿入されるための開口部を有し、
 前記第1培養槽が前記第2培養槽の開口部から、前記第1培養槽の底面に配された物質交換構造体がその槽に貯留された前記好気条件の培養液に浸漬するよう挿入され、
 前記物質交換構造体を除き、前記第2培養槽の内部が閉鎖状態となるように、当該第2培養槽と前記第1培養槽との間で開放されている部分を閉じた状態とし、前記第1培養槽と前記第2培養槽との接続部にガス非透過性の封止部材を備え、
 前記第2培養槽の頂面にある外部に開放されている部分を密閉するように設けられるガス透過性の保湿部材を備え、前記第1培養槽が嫌気条件下となる、培養システム。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • KYOTO UNIVERSITY
  • Inventor
  • KATAYAMA Takane
  • KAMBE Taiho
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JO JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
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