WO2009078637A2

WO2009078637A2 - Cell culture container and cell culture system

Info

Publication number: WO2009078637A2
Application number: PCT/KR2008/007404
Authority: WO
Inventors: Hyo Joo Song
Original assignee: Hyo Joo Song
Priority date: 2007-12-18
Filing date: 2008-12-15
Publication date: 2009-06-25
Also published as: KR20090065643A; WO2009078637A9

Abstract

These inventions are on the new cell culture container (instrument or flask or dish) and the new cell culture system for induced (guided or directed) differentiation and proliferation of target cells. One invention is new cell culture containers, one is filed (2 layered) and another is single. The shapes of containers are round or quadrangular shape. This container has the ports (for outflow and inflow) near the bottom of cell culture container, and the cap (or cover) has the germ barrier filter membrane permitting passage of air or gases. The quadrangular container has the dividing wall which separating inner space, and the wall has the filter permitting passage of culture media into opposite compartment. The another invention, new cell culture system, is composed of new cell culture container, peristaltic pump, filters, three way valves and connecting tubes. This system is designed to permit uni-directional flow of media and to culture the target cells in induced (guided or directed) proliferation or differentiation. The induced proliferation and differentiation in this system are under the influences of humoral factors and their sources are micro-environments or epigenetic factors such as organs, tissues or cells in containers. This new culture system separates the cultured target cells from the humoral factor sources and designed for easy pure collection of target cells which are undergone a induced proliferation or differentiation without mixture of humoral factor sources.

Description

CELL CULTURE CONTAINER AND CELL CULTURE

SYSTEM.

Technical Field

[1] The technical field, which these inventions belong, is biologic science. These inventions are designed to assist the better and efficient cell based treatment for treatment of incurable disease or disease difficult to cure, ultimately

[2] The new cell culture container (instrument or flask or dish) and new cell culture system using new cell culture container in these inventions are designed for induced (guided or directed) proliferation and differentiation of cultured target cells including stem cells using humoral factors secreted from micro-environments or epigenetic factors, and for easy collection of unmixed single kind of target cells efficiently in low cost.

[3]

Background Art

[4] The cells cultured in in vitro system show the various characteristics during proliferation and differentiation. Most cells tend to proliferate and differentiate after they have attached on the bottom of culture container (flask or instrument or dish) and they stop proliferation when the bottom of the culture container is completely occupied by proliferating cells. But some cells are continued to proliferate in multiple layer (stacked cell layer), sometimes form a mass attached on the bottom of culture container (flask or instrument or dish). Some cells are proliferating in suspension state in culture media. These inventions are designed for those cells which are proliferating and differentiating after attached on the bottom of culture container.

[5] For the culture of the cell, cell culture container (flask or instrument or dish) and many other types of equipment are involved. Among these, the most important for the culture of the cell is container (flask or instrument or dish) where cells are growing. The cell culture containers (or instruments) are shaped in dish (culture dish), flask (culture flask), bottle (roller bottle), slides (chamber slides) or specific form according to their purpose, such as modified Boyden chambers. A few people used the cell culture container which is modified for their purpose. Basically, the common types of culture containers are dish type, flask type or roller bottle type.

[6] Modified Boyden chamber is different from conventional culture container system.

The modified Boyden chamber has upper chamber and lower chamber, and the bottom of the upper chamber has the membrane which has the many micro-pores. Through these micro-pores, fluid (media) in both upper and lower chamber can communicate each other. This culture system is used for specific purposes, for example, study on invasion or metastasis or motility or differentiation etc.

[7] When we use the modified Boy den chamber, for the upper chamber, we seed the target cells which we want to study. And specific substances are put into lower chamber. Through the micro-pores in upper chamber bottom membrane, the target cells in upper chamber shows some responses influenced by specific lower chamber substances. The communication of fluid between upper and lower chamber in modified Boy den chamber is the very important. But the significant defect of this communication appeals when lower chamber substances were cells of another type of tissue or organ. If we culture different cells in upper and lower chamber in modified Boyden chamber, at the end of experiment, those different cells are exist in mixed state at the lower chamber. And when we try to separate and to collect the specific upper or lower chamber cells among mixed cells, there is significant difficult and sometime result failure of the experiments. For collection of the specific cells among mixed cells, it is very difficult and need very expensive costs. For example, when we plan to collect the upper chamber target cells and plan to use them for cell based therapy among mixed cells, it will cost very much, and usually impossible for clinical application.

[8] Differentiation of cultured cell is highlighted recently due to stem cell study. There are lots of studies related with stem cell differentiation and this differentiation study cost very much. The goal of stem cell study is to get the sufficient number of cells which are enough for cell replacement therapy in minimal differentiation, and induce this minimally differentiated stem cell to differentiate into the cell type we wanted (induced or guided or directed differentiation). If this goal is accomplished, it would be the accomplishment of customized medicine.

[9] The characteristics of stem cell are self -renewal and multi-potency in differentiation.

The multi-potency in differentiation is the regarded as an essence of the hope for the treatment of disease which is incurable or difficult to cure. Stem cell differentiation into the cell or tissue type as we wanted would be the first step we can treat the disease. And, present condition, the most common methods for the differentiation of stem cell into the cell type what we want, would be addition or supplement of the some growth factors into the stem cell culture media or genetic manipulation of the stem cell.

[10] The addition or supplement of growth factors into cell culture media cost very much, because we do not know the specific growth factors for specific differentiation. Some growth factors get the reproducibility in differentiation but many of them do not.

[11] The dilemmas in stem cell study are two. One is lack of enough numbered minimally differentiated stem cells and another is induction of cell differentiation into what we wanted. Right now, there is much progress in expansion of stem cell at minimally differentiated state. But the induction of differentiation into cell type, which we plan, remained much to be studied. Especially, when we try to differentiate the stem cell into the cell type we wanted in low cost, it is impossible at present time. For this reason, the invention or development of new instruments, method or system for induction of stem cell differentiation into cell type we wanted is very important for treatment of incurable disease or disease which is difficult to cure under the based on cell.

[12] Recently, niche is very important topic related with stem cell differentiation. And the values of the micro-environment or epigenetic factor appeared as the important factor in stem cell differentiation, too. Cur poor knowledge on the growth factors which are necessary for the differentiation of stem cell into specific cell types make people to mention these specific growth factors as humoral factors or paracrine factors.

[13] Micro-environments and epigenetic factors related with cell differentiation are focused recently. So, for the study of micro-environments or epigenetic factors related with differentiation, modified Boyden chamber and co-culture system become conspicuous. But, the critical defect in these two conventional systems, modified Boyden chamber and co-culture system, is mixture of different cell types, and the pure collection of target cells in single type among mixture of different cells, what we wanted, is very difficult and hampered by their nature.

[14] In modified Boyden chamber, if target cells, including stem cells, are seeded in upper chamber and the lower chamber is seeded with some other conventional cell or tissues which are regarded as the source of humoral factors, the target cells in upper chamber will move into lower chamber during incubation through the micropores at upper chamber bottom and some will have differentiation. The collection of the pure target cells, which would be affected by lower chamber micro-environment or epigenetic factors, would be almost impossible in normal living state of stem cell. And, for cell based therapy, we cannot use these mixed differentiated cells.

[15] In co-culture system, more than two types of different cells are cultured in a same culture system and they have cellular communications each other and grow in mixed condition. The goal of co-culture system in proliferation and differentiation are obtaining some positive or negative effects from the cell to cell communication. Qinical aspect, co-culture has the very significant defect, difficulties to collect cell in pure typed cells among mixed ones.

[16] There is the urgent need to solve these problems which resided in modified Boy den chamber and co-culture system which using micro-environment or epigenetic factors for the rapidly developing cell based treatment. These inventions, the new cell culture containers (instruments or flask or dish) and cell culture system are designed for the efficient, rapid and low cost collection of the target cells among induced (guided or directed) proliferated or differentiated mixture of cells to solve the defects of modified Boyden chamber or co-culture system.

[17]

[18] Following references provide the scientific backgrounds on these inventions :

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Y, ding M. Deng H. Directed differentiation of human embryonic stem cells into functional hepatic cells. Hepatology 45:1229-1239, 2007.

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Mudipalli A, Megan R, Ip MM. Mammary fibroblasts stimulate growth, alveolar morphogenesis, and functional differentiation of normal rat mammary epithelial cells. In Vitro Cell Dev Biol Animal 36:578-592, 2000.

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Langerhans cell precusors. Eur J Cell Biol 83:805-811, 2004.

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Chondrogenic differentiation of human bone marrow stem cells in transwell cultures: Generation of scaffold-free cartilage. Stem Cells 25:2786-2796, 2007.

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In vitro differentiation of human cord blood-derived unrestricted somatic stem cells towards an endodermal pathway. Cytotherapy 9:362-379, 2007.

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Re⁰I H, Gabriel C. Dose dependent immunomodulatory effect of human stem cells from amniotic membrane: a comparison with human mesenchymal stem cells from adpose tissue. Tissue Eng. 13:1173-1183, 2007

[65] Disclosure of Invention Technical Problem

[67] For the cell based treatment of incurable disease or disease that is difficult to cure, preparation of the target cells, which are proliferated or differentiated as we want (induced or guided or directed proliferation or differentiation), is extremely important. For the induced (guided or directed) proliferation and differentiation using humoral factors secreted by micro-environment and epigenetic factors are very important. Modified Boyden chamber and co-culture system are most common form of cell culture systems utilizing humoral factors. But, both modified Boyden chamber and co- culture system have critical defect for cell based treatment that these systems do not separate the target cells from humoral factors sources.

[68] And the collection of the uniform and one kind of cells which are proliferated and differentiated as we wanted, is very important for proper treatment of cell based treatment. In cell based treatment with mixed cells which are unnecessary for target disease treatment, unnecessary cells can cause unexpected significant events.

[69]

[70] Conventional culturing systems for induced (guided or directed) proliferation or differentiation are modified Boyden chamber or co-culturing system. The basic mechanism for these systems are using humoral factors for induced proliferation or differentiation. And the humoral factors are closely related with micro-environment and epigenetic factors.

[71] The technical problems of these culturing systems are extreme difficulties in collection of the pure one kind of target cells, which do not mixed with humoral factor sources.

[72]

Technical Solution

[73] The induced (guided or directed) proliferation or differentiation of target cells and collection of these cells without contamination of other cells are absolutely necessary for the cell based therapy which is regarded as the future solution of incurable disease or disease difficult to cure.

[74]

[75] These inventions are designed to solve the significant defects that is very difficult and practically impossible to collect the target cells in pure one kind of cells in conventional system (modified Boyden chamber or co-culture system) for induced (guided or directed) proliferation and differentiation. To achieve this goal, collection of one kind of target cells with high efficient, easy in method and low cost for induced (guided or directed) proliferated or differentiated target cells, these inventions are designed for complete separation of target cells (including stem cells) from humoral factor sources, by new cell culture container (instrument or flask or dish) and new cell culture systems involving the new cell culture containers.

[76] Because the target cells, which are in induced proliferation or differentiation, are located at separated compartment (container or chamber) from humoral factor sources, the collection of the pure one kind of cell is very easy

[77]

[78] For detailed explanation on these inventions, following descriptions on the structure amd mthod for using these inventions are informative.

[79]

[80] These inventions are composed of two inventions which are mutually involved. One is new cell culture container (instrument or flask or dish) (figure 1 ) and another is new cell culture system using new cell culture container. The new cell culture container (instrument or flask or dish) and new cell culture system are closely related with humoral factors which are secreted from micro-environments and epigenetic factors, such as organ, tissue or cells. The new cell culture container (instrument or flask or dish) in these inventions separate target cells (stem cells or other conventional cells) from cells of humoral factor sources which would be related with micro-environmental or epigenetic factors.

[81] Another invention, new culture system (figure 10 and 11 in drawing) that is using this new cell culture container, is system for induced (guided or directed) proliferation or differentiation of target cell (including stem cell). The new cell culture system is composed of new cell culture containers, peristaltic pump, filters, three way valves and connecting tubes. The new cell culture system, permit target cells to contact with the humoral factors, which are secreted by the micro-environments and epigenetic factors, for proliferation and differentiation and lead the induced (guided or directed) differentiation of the targeted cells for cell based therapy. But this system excludes direct contact between targeted cells from humoral factor sources, the micro-environments and epigenetic factors.

[82]

[83] Figure 1 is the representative drawing of new cell culture containers (instrument or dish) and, figures 10 and 11 are on new cell culture systems. [84]

[85] Figure 1 is the sectional views of the two storied cell culture container (instrument or flask or dish) (AA) which filed up two single storied cell culture container and single storied cell culture container (BB).

[86] The two storied cell culture containers (instrument or dish) (AA) have upper chamber and lower chamber, which tightly closed each other. This figure is the just looks likes the filed two single storied new culture containers (instrument or dish) but adhesion (connecting) part (B) of two cell culture instruments show that the lower chamber do not have a cap (cover or envelop) and the upper part of the lower chamber wall has the screw shape (figure 3 F) which fit for the screw, which made at the lower extended wall of upper chamber below the bottom (figure 3 F), to fasten upper and lower chambers water and air tightly. The tightly adhesive closed two chambers, upper and lower chambers, do not permit passage of any gas (air) or water through the fitted connection portion.

[87]

[88] Both upper and lower chamber have the ports at two sites opposite each other (figure

3 and 4). None have been definitely decided for inflow or outflow ports. Inflow or outflow will be decided according to the flow direction made by operating peristaltic pump.

[89]

[90] The ports are located at the height that the cells, which occupy the bottom of the each cell culture containers (instrument or dish) (confluence or in some cases of sub- confluent cultured cell), could not climb in the contact inhibition state. This will prevent the losing of the targeted cell or cells of humoral factor source, into outside of ports. But the ports locate within the upper surface of the media.

[91]

[92] Figure 3 is the enlarged drawing of the connecting part between upper chamber and lower chamber (AA in figure 1) area. Inner side of the downward extended side wall of the upper chamber bottom is designed for furrow and the outer side of the upper part of the lower chamber is designed for projection. The furrow and projection are designed to fit completely. If, the upper chamber and lower chamber are closed in tight adhesion, one set of two storied cell culture containers (instrument or dish) are formed. D is the enlarged drawing of the ports. The outer surface of the ports is made of saw tooth like shapes to prevent the connecting tubes easily slipping off. [94] Figure 4 is the enlarged lower chamber (C) near the bottom. The inflow (or outflow) ports (connecting parts) are equipped with mechanism, such as saw like shape, which the fixed connecting tubes do not easily slip out. The ports (inflow or outflow ports) are situated just above the bottom of the cell culture containers (instrument or dish) and the ports are designed with the shape (just like saw tooth shape) to prevent the connecting tube comes out from the ports.

[95]

[96] Simultaneously, the enlarged part of cap (cover or envelop) of two storied (A in figure 1) or single storied cell culture containers (instrument or dish) (K in figure 1), as in figure 2, have the screw shaped ragged figures or similar mechanisms at inside of the wall which fit completely with the upper part of the wall of upper chamber in two storied or single storied new cell culture containers (instrument or dish). Through this cap site (figure 2), nothing could pass, including water or gas (including oxygen or air). But this invention, new cell culture containers (instrument or dish) had the germ barrier filter on the cap (E) or side wall (E) of the upper or lower chamber, as figure 6, which permitting passage of the gases( air, oxygen or carbon dioxides so on). This germ barrier filter membrane has the micro-pores which limiting passage of the germs (bacteria or so on) but permitting passage of gases. So, the oxygen in the incubator could diffuse into culture media without problem. The targeted cells or humoral factor sources cultured within these cell culture containers (instrument or dish) are cultured in germ free condition if the cultured cell or culture media are not infected, initially. In any cases, two storied or single storied one, if the germ barrier filter membranes are installed at the cap, the germ barrier filter membranes at side wall of the containers (instrument or dish) could be omitted.

[97]

[98] Single storied cell culture containers (instrument or dish) (BB in figure 1) is just same as the upper chamber, which does not have the connecting mechanism with lower chamber, of the two storied new cell culture containers (instrument or dish). The cap (X) (cover or envelop) and cell culture container have tight closing mechanism prevent air and water passage. On the cap, there is germ barrier filter membrane which has the micro-pores permitting air or other gases but excluding the passage of the germs including bacteria etc. (figures 6 and 8). The germ barrier filter membrane can be placed at the side wall of the new cell culture containers (instrument or dish) according to manufacturer's design.

[99] [100] Figure 9 is the top side view of the quadrangle type cell culture containers

(instrument). There is a partition (qq-qq) at inside of regular or rectangular shaped cell culture containers (instrument). Membrane filter is at the partition. The membrane filter has many micro-pores permitting free passage of media with humoral factors which are secreted from micro-environments or epigenetic factors placed over the membrane filter. The micro-pores are installed above the height of confluent or sub- confluent cells can not reach. And cap (or cover) of the quadrangular shaped cell culture containers (instrument) had the germ barrier filter membrane (Z) for diffusion of the air or other gases into media. When the cap (or cover) of these quadrangular type cell culture containers (instrument) are closed adhesion tightly, oxygen, air or other gas will diffuse only through the membrane (at cap or cover) into media. For the media change during cell culture, the cap (or cover) of these quadrangular type cell culture instruments (containers) should be opened.

[101]

[102] Figure 10 is the new cell culture system using two storied new cell culture container (instrument or dish) in closed circuit. For completion of this new cell culture system, two storied new culture container in this invention, peristaltic pump for uni-directional flow of media, filter of various sized pores for prevention of passage of cell or cellular debris into ahead chamber where target cells or humoral factor sources are growing, three way valves for flow direction change, and connecting tubes. The humoral factor sources, organ or tissue or cells which have nature of micro-environments and epigenetic factors, are located at the one new cell culture container (one of upper or lower chamber). And the targeted cells, including stem cells in occasion, are located at another new cell culture container. As mentioned before, flow of the media is unidirectional and media change will be processed by three way valve control. Properly adjusted three way valve will turn the media flow direction from closed circuit to other direction to provide an outflow of old media into outside vessel. And reversely, differently adjusted other three way valve provide aspiration of the new fresh media from the outside of sterile media container into each new cell culture container (instrument or dish). The later case, the aspirated media can be moved into chamber after filtered in this circuit.

[103]

[104] The media containing humoral factors which secreted from micro-environment or epigenetic factor sources would circulate via connecting tubes and enter into the new cell culture container (instrument or dish) where targeted cells are growing, after the filtering cell or cell debris. The humoral factors will induce the differentiation of target cells into cell types what we want. So, induced (guided or directed) differentiation is accomplished. At the end of culture of target cell, after separation of the upper chamber and lower chamber, the induced (guided or directed) differentiated target cells can be purely collected without contamination from other cells which secreted humoral factors.

[105]

[106] Figure 11 is the new cell culture system using single storied new cell culture containers (instrument or dish). New cell culture containers (G and H) are placed at separated sites. The media from the one new cell culture containers (for example G)(where target cells including stem cells are growing) outflow port (Gaa) moved via the connecting tube D and passed three way valve and passed peristaltic pump and passed filter and moves into another new cell culture instrument (container) H through inflow port Hdd. The media from new cell culture containers H, which contains humoral factors obtained from the micro-environment or epigenetic sources (organ or tissue or cells culturing at H) pass outflow port (Haa) and connecting tube (tube C) and pass the filter, and pass the peristaltic pump and three way valve and flow into the inflow port of new cell culture container G (Gdd). So, the media, from the chamber where targeted cell are grow, flow into other chamber and obtain the humoral factor from micro-environment or epigenetic factor sources and return into target cell chamber and induce (guide or direct) differentiation under the effects of humoral factors.

[107]

Advantageous Effects

[108] The advantages of these inventions, new cell culture container (instrument or flask or dish) and new cell culture system, designed for cell preparations which will be used in cell based treatment are two. First, these inventions permit the preparation of induced (guided or directed) proliferation or differentiation of target cells by using humoral factors related with micro-environment or epigenetic factor as conventional cell culture systems. And second, easy, efficient, cost effective and labor saving collection of the target cells in pure one kind of cells, without mixing of humoral factor sources with efficiency and low cost.

[109]

Brief Description of the Drawings [110] The figures in these inventions are 11 and figures 2 to 4 and 6 are enlarged drawing of above figure 1. [I l l] [112] Figure 1 is the sectional views of the two storied cell culture container (instrument or dish) (AA) which filed up two signle storied cell culture container and single storied cell culture container (BB). Related with figure 1, detailed stories are mentioned before at the disclosure section (subsection of technical solution). [113] [114] Figure 2 is the enlarged drawings of mechanisms related on the cap area of cell culture containers (instrument or dish). This drawing shows that the mechanism is designed to close in tight adhesion to prevent passage of air or water etc. So, tightly close the cap prevents passage of gas or water. [115] [116] Figure 3 are enlarged drawing in connection part between upper chamber and lower chamber in two storied cell culture container (instrument or flask or dish) and enlarged drawing of ports (outflow and inflow), [117] [118] Figure 4 is the enlarged detailed figure 1 on the bottom of two storied cell culture containers (AA) or single storied cell culture instruments (BB) which have ports. As enlarged drawing of CA, the ports having saw tooth shape prevent easy spill connecting tube off. [119] [120] Figure 5 is a enlarged perstaltic pump part of new culture system. This figure shows peristatic pump with connecting tubes. The peristaltic pump is designed to have 2 or more wings (ee). The shape and size of the each wing should be exactly balancing.

This peristaltic pump pushes the culture media in elastic connecting tube intermittently into one direction forming uni-directional flow. [121] [122] Figure 6 is germ barrier filter membrane in cap or side wall of cell culture containers

(instrument or dish) which has multiple micro-pores which are permitting passage of air or other gases but preventing any passage of germinal organisms. [123] [124] Figure 7 is side view of the germ barrier filter membrane installed two storied cell culture instrument, kk, gg, oo and mm are ports (inflow or outflow). [125] [126] Figure 8 is the top side view of the round shaped cell culture instruments. The germ barrier filter membrane is placed at the cap of containers (instrument or dish) which permitting passage of air or other gases but inhibiting any kind of germs.

[127]

[128] Figure 9 is top side view of the rectangular shaped container (instrument or flask)

[129]

[130] Figure 10 is general view of linking in new culture system that composed of a two storied cell culture container (instrument or flask or dish), peristaltic pump, filters, three way valves and connecting tubes,

[131]

[132] Figure 11 is general view of linking in new culture system that composed of two single storied cell culture container (instrument or flask or dish), peristaltic pump, filters, three way valves and connecting tubes,

[133]

Best Mode for Carrying Out the Invention

[134] These inventions are composed of two inventions, one is new cell culture container (instrument or flask or dish) and another is new cell culture system using new cell culture container (instrument or flask or dish).

[135]

[136] One invention is the new cell culture container (instrument or flask or dish) (drawing 1) which is round shape (culture dish type), or regular square or rectangular type. Culture dish type round new cell culture container have two ports (outflow and inflow) near the bottom of each container, and had the cap (or cover) which has the germ barrier filer membrane excluding passage of germs but air or gases. The round culture dish types divide into two types, one is two storied new cell culture container which tightly adhered two instruments without lower chamber cap or envelop. Another is single storied cell culture container which is separated each other. All new cell culture containers in round dish shape equipped two ports oppositely located in each container. The regular square or rectangular new cell culture containers have the partition (dividing wall) separating inner square into two and the partition has the membrane which have many micro-pores permitting passage of media but excluding passage of cell or cell debris.

[137]

[138] Another invention is new cell culture system (drawing 10 and 11). The new culture system are composed of new cell culture container (instrument or flask or dish), peristaltic pump, filters, three way valves, and connecting tubes. This invention separate the humoral factor sources(organs or tissues or cells for micro-environments or epigenetic factors) from the targeted cells which we want to induce (guided or directed) the proliferation or differentiation. This invention separate the target cells, which we want to collect, from the humoral factor sources, such as micro-environments or epigenetic factors (organ, tissues or cells). But the media containing humoral factor for induced (guided or directed) proliferation or differentiation will circulate from one chamber (new cell culture instrument or container) to another where target cells are culturing, but the cells in both chamber will not meet because the filters in this invention (new culture system) prevent passage of the cells or cellular debris. So, the mixing of the cells in these two separated chamber is basically excluded and we can collect pure targeted cell without contamination.

[139]

[140] For application examples, the two storied cell culture container (instrument or flask or dish) are divided into upper chamber or lower chamber. And in single storied cell culture instruments (containers), two cell culture instruments are designated as left chamber or right chamber.

[141] The composition of the new culture system is as follow. The new culture system is designed to form a closed unidirectional circulation of media. From the outflow port of one (first) chamber, the media flow through the connecting tube, and pass the filter and three way valves. The soft connecting tube passes the peristaltic pump and connected to the inflow port of another (second) chamber. The culture media, from the outflow port of another (second) chamber, flow through the connecting tube (where filter and three way valve are interposed).

[142]

[143] In new cell culture system using two storied cell culture container (instrument or dish), the appropriate volume of media with targeted cells or humoral factor sources at lower chamber should be prepared before the adhesion tight closure (air and water tight closure) between upper and lower chambers.

[144]

[145] At practical application example using these inventions (new cell culture container and new culture system) is as follow. In this example, the target cell for proliferation and differentiation is the adipose tissue derived stem cells (ADSCs) and the humoral factor source is tiny chopped bladder muscle tissues in dog. Two kinds of cell or tissues are prepared separately according to known protocol. The target cells are stem cells collected from subcutaneous fat tissue and other humoral factor sources are chopped urinary bladder wall muscle of same dog.

[146]

[147] About 12 Kg weighted adult dog is pretreated with atropin and anesthetized with pentobarbital and zylazine. After shaving, antiseptic painting and incision, subcutaneous fat blocks and parts of urinary bladder are removed and keep them in cold DMEM media in sterile condition. Using each specific protocol, the adipose tissue derived stem cells (ADSCs) and chopped tiny urinary bladder muscle tissues are collected in sterile condition.

[148]

[149] After the preparation of new culture system including filling of media in tube and new cell culture containers (instruments or flasks or dishes) have finished under the sterile condition, the target cell ADSCs are seeded onto new cell culture container (instrument or flasks or dish) upper surface of bottom in upper chamber or left chamber (first chamber). And the chopped urinary bladder muscle tissues are placed onto new cell culture containers (instruments or flasks or dishes) upper surface of bottom in lower chamber or right chamber (second chamber). If cap (cover or envelop) and connecting part between upper and lower (left or right) chambers have air and water tightly closed in new cell culture containers, the operation of peristatic pump begin after 12 to 24 hours to allow the attachment of cells (or tissues) on the surface of the each chamber bottom. The speed of the pump is decided depend on the size of the new cell culture containers. And cell cultures are maintained according to scheduled plan.

[150] Diffusion of oxygen and carbon dioxide into media will be made naturally through the germ barrier filter membrane in caps or side wall of the new cell culture containers (instruments or flasks or dishes). The media change can be accomplished by proper operation of the three way valves after connect the sterile tube into one port of three way valves. Drain the old media into outside vessel and aspiration of new media into chamber are easily made with manipulation of valves, turn the flow direction.

[151] If we apply the new cell culture system with two storied cell culture instruments in drawing 10, the circuit is as follow. After seeding of the target cell, ADSCs, into the upper chamber and placement of chopped urinary bladder muscles at the lower chamber, the two chambers are connected air and water tightly (F) and cap (envelop) has closed tightly. Then, the peristaltic pump is operated after 12 to 24 hours to allow the attachment of cells (or tissues) on the surface of the each chamber bottom. The media from the upper chamber move through outflow (gg) and flow along the connecting tube A and cellular debris will be filtered being passing the filter (XXX). The filtered media pass the peristaltic pump and pass the three way valve (xx) and filtered media enter the low chamber inflow port (oo). The media acquired humoral factors during passing the humoral factor sources, chopped urinary bladder muscles, pass the outflow port of the lower chamber (mm) and circulate through the connecting tube B and pass the three way valve (yy) and passed peristatic pump and filter the tissue debris at filter (YYY) and filtered media containing humoral factors flow into inflow port of upper chamber (kk). So, the targeted cells in upper chamber and humoral factor source of lower chamber are physically completely separated but the humoral factors from lower chamber can affect on the targeted cells in upper chamber. These systems indicate the adaptation of the merits of modified Boyden chamber or co-culture system but prevent their critical defect, mixing of the different clones of cells.

[152] Similar application of new cell culture system with single storied new cell culture container (instrument or flasks or dish) is shown at draw 11. Both new cell culture container (instrument or flasks or dish) G and H separated each other. If targeted cells (ADSCs) are seeded at one cell culture instrument (first) G, the media from container G pass the outflow port (Gaa) and flow through the connecting tube D and pass the three way valve (Gbb) and then pass the peristaltic pump and filter (Gcc), and flow into chamber where humoral factor sources (chopped bladder muscle cells) are placed, another cell culture instrument (second) H, through inflow port (Hdd). The media acquire humoral factors from the humoral factor sources, (chopped urinary bladder muscles) pass the outflow port of chamber H (Haa) and flow through connecting tube C and passed filter (Hcc) and pass the peristaltic pump and three way valve (Hbb) and and enter chamber G through inflow port of chamber G (Gdd).

[153]

[154] The advantages of new cell culture system are; first, the new culture system permit the indirect biological communication between targeted cells and humoral factor sources through circulating conditioned media containing humoral factors and this communication lead the induced (guided or directed) proliferation or differentiation of target cell. And, control of the differentiation state of target cell will be possible, with data analysis after repeated experiments. Second, this system is tightly closed system. So, there is no room for contamination of germ, and the cost down is achieved in induced cell culture. And the media changes can be done easily in this closed cir- culation circuit by operation of three way valve, easily. Third, collection of the uncon- taminated targeted cells which are induced (guided or directed) proliferated or induced differentiated cells for cell based therapy, very easily.

[155]

Industrial Applicability

[156] These inventions, the new cell culture containers (instruments or flasks or dishes) and new culture systems are applicable for research fields in the laboratory experiment and in the field of bio- industry or hospital. The much expanded number of cells that are induced (guided or directed) proliferated or differentiated targeted cells which will be used for cell based therapy. Because these inventions preclude contamination from unwanted cells in proliferated and differentiated target cells, it will reduce the production cost of target cells and medical expanses.

[157]

Claims

[1] In round cell culture container (instrument or dish),

On the side wall of cell culture container, at a height higher than which the confluent cells can not reach from upper surface of the bottom of the cell culture container, exist ports for inflow or outflow of cell culture media.

The cell culture container has a cap (or a cover) which has air and water tight adhesive closing system.

The cap (or cover) has a germ barrier filter membrane which permits passage of air or other gases and excludes passage of any kind of germ.

[2] In above claim 1, two storied cell culture container is piled up to forms one unit.

When the upper cell culture container (upper chamber) and lower cell culture container (lower chamber) are connected, the connecting parts are completely air and water proof.

The upper chamber and the lower chamber each have two ports for inflow and outflow of the culture media.

Side wall of the upper chamber and the lower chamber can have another germ barrier filter membrane which exclude any kind of germinal passage but permitting passage of air or other gases.

[3] In quadrangular type cell culture container (instrument or flask), the quadrangular type cell culture container has a wall which divides the inner space into two, the dividing wall has a micropore membrane which permits passage of media only and not permitting any cell, tissue or cell debris.

On the cap (or cover), there is a germ barrier filter membrane which permits passage of air or other gases but excludes passage of any kind of germ.

[4] The inventions of new cell culture system is composed of cell culture containers

(instrument or flask or dish) that are mentioned in claim 1 and claim 2 and peristaltic pump and filters and three way valves and connecting tubes. This cell culture system is air and water proof, which does not permit the invasion of germs but permit passage of air or gases only through germ protecting filter membrane in the cap (or cover) of cell culture container. The filter in this cell culture system allows free passage of media flow, which contains humoral factors, but prevents passage of cell or tissue and their debris. This cell culture system achieves media change by operation of three way valve handles, which drain the old media through a three way valve port and aspirate fresh media through another three way valve port.