WO1990015859A1 - Dynamic flow cell culture system - Google Patents

Dynamic flow cell culture system Download PDF

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Publication number
WO1990015859A1
WO1990015859A1 PCT/US1990/000480 US9000480W WO9015859A1 WO 1990015859 A1 WO1990015859 A1 WO 1990015859A1 US 9000480 W US9000480 W US 9000480W WO 9015859 A1 WO9015859 A1 WO 9015859A1
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WO
WIPO (PCT)
Prior art keywords
liquid
divider member
conduit
chamber
gas
Prior art date
Application number
PCT/US1990/000480
Other languages
French (fr)
Inventor
Hagop M. Kantarjian
Original Assignee
Board Of Regents, The University Of Texas System
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Filing date
Publication date
Application filed by Board Of Regents, The University Of Texas System filed Critical Board Of Regents, The University Of Texas System
Publication of WO1990015859A1 publication Critical patent/WO1990015859A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/34—Internal compartments or partitions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/44—Multiple separable units; Modules

Definitions

  • the present invention relates to devices for culturing cells.
  • bone marrow transplantation In that procedure, bone marrow is harvested from a leukemia patient and is cultured in vitro under conditions that favor the growth of normal cells but cause leukemic cells to die out. The end result is a culture which should be substantially free of cancerous cells. The normal cells are recovered and transplanted back into the patient.
  • the present invention concerns a cell culture device which includes a bottom plate; an upper enclosing member; a side enclosure which, together with the bottom plate and upper enclosing member, defines a completely enclosed volume; at least one horizontal divider member which is located between the bottom plate and the upper enclosing member and is attached around its perimeter to the side enclosure, thereby dividing the enclosed volume into at least two chambers, one of which is an upper chamber and one of which is a lower chamber; at least one liquid conduit traversing each horizontal divider member, the liquid conduit having a top opening and a bottom opening, with the liquid conduit extending upward above the horizontal divider member so that its top opening is located in the chamber above the horizontal divider member at a selected height, with the liquid conduit also extending downward below the horizontal member so that its bottom opening is located in the chamber below the horizontal divider member at a selected height; at least one gas conduit traversing
  • the bottom plate, upper enclosing member, and each horizontal divider are preferably flat plates which are parallel to each other.
  • the side enclosure preferably is a number of flat vertical plates which are perpendicular to the bottom plate.
  • the device can also include at least one vertical support post which extends between the upper enclosing member and a horizontal divider member, between a horizontal divider member and the bottom plate, or between two horizontal divider members.
  • the inlet and outlet openings preferably include a liquid inlet and a gas inlet, both of which connect with the upper chamber, and a liquid outlet, which connects with the lower chamber.
  • a cell culture device in accordance with the present invention includes a flat bottom plate; a flat upper plate; four rectangular side walls which are joined to each other and to the bottom plate and upper plate, thereby defining a completely enclosed volume; an upper divider member which is a flat plate parallel to the bottom plate and the upper plate and located inbetween them, and which is joined around its perimeter to the side walls; a lower divider member which is a flat plate parallel to the bottom plate and the upper plate and located inbetween the bottom plate and the upper divider member, and which is joined around its perimeter to the side walls, whereby the enclosed volume is divided into an upper chamber, a middle chamber, and a lower chamber, each of which has approximately equal volume; at least one upper liquid conduit traversing the upper divider member, the upper liquid conduit having a top opening and a bottom opening, with the upper liquid conduit extending upward above the upper divider member so that the top opening is located in the upper chamber at a selected height, the upper liquid conduit also extending downward below the upper divider member so that its bottom opening
  • the present invention also relates to a dynamic flow cell culture system, which includes a cell culture device as described above; a liquid feed conduit which connects at one end to the cell culture device and connects at the other end to a supply of liquid needed for cell culture, and supplies a flow of the liquid to the device; a gas feed conduit which connects at one end with the cell culture device and connects at the other end to a supply of gas needed for cell culture, and supplies a flow of the gas as needed to the device; and a liquid drain conduit which connects at one end with the cell culture device, and drains a flow of liquid from the device.
  • at least one additional cell culture device as described above is stacked on top of another one, allowing the overall volume to be increased without taking up additional floor space.
  • a dynamic flow cell culture system in accordance with the present invention, cells to be cultured are introduced into a sterilized cell culture device along with a suitable liquid growth medium.
  • the device is then supplied with an atmosphere which consists essentially of air plus approximately 5% C0 2 , and the cells are incubated. After a sufficient time has passed, the contents of the device can be removed, either through the liquid drain conduit, or through the liquid feed conduit after inverting the device.
  • the present invention provides a semi-closed system for cell culture which occupies less space and requires less effort than prior systems. Further, the present invention reduces the risk of contamination by eliminating the need for the cell culture device to be opened during the culturing process.
  • the present invention is useful for purging tumor cells from the bone marrow of patients who have diseases such as chronic myeloid leukemia, acute myeloid leukemia, acute lymphocytic leukemia, myeloidplastic syndrome, and lymphoma. It is also useful for a variety of other cell culture operations.
  • Fig. 1 is a perspective cut-away view of a cell culture device in accordance with the present invention.
  • Fig. 2 is a top view of a cell culture device in accordance with the present invention.
  • Fig. 3 and Fig. 4 are side cross sectional views of the device shown in Fig. 2, taken along the axes designated 3-3 and 4-4 in Fig. 2, respectively.
  • Fig. 5 shows a side cross sectional view of a cell culture device in accordance with the present invention while in operation.
  • Fig. 6 shows a dynamic flow cell culture system in accordance with the present invention.
  • the structure of a specific embodiment of a cell culture device in accordance with the present invention is shown in Figures 1-4.
  • the device 10 has flat exterior surfaces with a rectangular cross-section.
  • the device 10 has an upper plate 12, a bottom plate 14, and four side walls 16, which together define a completely enclosed space.
  • the only openings in these surfaces and walls are a liquid inlet 18, a gas inlet 20, and a liquid outlet 22.
  • the device 10 also has two internal horizontal dividers, which are an upper divider member 24 and a lower divider member 26. These two dividers, in conjunction with the upper plate 12 and bottom plate 14 and the side walls 16 define three separate enclosed chambers: an upper chamber 28, a middle chamber 30, and a lower chamber 32.
  • These three chambers have fluid communication means that will be described below.
  • the liquid inlet 18 and gas inlet 20 connect the upper chamber 28 with the outside of the device, while the liquid outlet 22 connects the lower chamber 32 with the outside.
  • Each of the upper plate 12, bottom plate 14, upper divider member 24, and lower divider member 26 are horizontal and are parallel to each other. Opposite side walls 16 are vertical and parallel to each other.
  • Four support posts 40 are provided which effectively extend from the upper plate 12 to the bottom plate 14, and provide additional structural support for the device 10. These support posts 40 can be hollow cylinders which extend through circular holes cut in the upper divider member 24 and the lower divider member 26. However, it is preferred to construct each support post 40 out of three separate cylindrical members 40A, 40B, and 40C.
  • the cylindrical member 40A extends vertically between the upper plate 12 and the upper divider member 24.
  • the other two cylindrical members 40B and 40C extend in the same way between the upper divider member 24, the lower divider member 26, and the bottom plate 14.
  • the top view of the device 10 shown in Fig. 2 illustrates the position of the fluid communication means between the three chambers 28, 30, and 32.
  • the four support posts 40 are evenly spaced in order to provide the maximum structural support.
  • a set of gas conduits 46 in the center of the device 10 is a set of gas conduits 46.
  • Fig. 4 is a side cross-sectional view of the device 10 taken along the axis designated 4-4 in Fig. 2. Looking from this side view one sees the support posts 40A, 40B, and 40C. On either side are lower liquid conduits 44 which connect the middle chamber 30 and the lower chamber 32. These conduits 44 preferably are hollow cylindrical tubes and are fixed in place in openings in the lower divider member 26. The openings in the member 26 should be no larger than necessary to insert the channel 44 and should have the same horizontal cross-sectional shape, so as to prevent fluid from flowing from one chamber to another chamber below it by seeping between the outside of the cylindrical tube and the divider member. Above and in the background can be seen upper liquid conduits 42.
  • Fig. 3 is a side cross- sectional view of the device 10 taken along the axis designated 3-3 in Fig. 2, one can again see a lower liquid conduit 44 which connects the middle chamber 30 and the lower chamber 32. One also sees two upper liquid conduits 42 which connect the upper chamber 28 and the middle chamber 30, in the same manner as described above for the lower conduits 44.
  • the set of gas conduits includes an upper gas conduit 46A and a lower gas conduit 46B.
  • the upper gas conduit 46A connects the upper chamber 28 and the middle chamber 30, while the lower gas conduit 46B connects the middle chamber 30 and the lower chamber 32.
  • Each component of the device including the upper plate 12, bottom plate 14, side walls 16, upper divider member 24, lower divider member 26, support posts 40, liquid conduits 42 and 44, and gas conduits 46A and 46B, are preferably transparent. Suitable materials include plastic and glass.
  • Operation begins by sterilizing the device 10, and then charging liquid, such as bone marrow in appropriate liquid media, through the liquid inlet 18.
  • liquid spills into the upper chamber 28, .and the liquid level in that chamber rises as more liquid is charged.
  • the liquid level reaches the top of the upper liquid conduits 42, it begins to spill through those conduits into the middle chamber 30.
  • the liquid level rises in the middle chamber 30 until it reaches the top of the lower liquid conduits 44. At that point, the liquid begins to spill through the lower liquid conduits 44 into the lower chamber 32.
  • liquid can be evenly distributed to all three chambers. Evenness of distribution is enhanced by locating the liquid conduits 42 and 44 in a regular pattern across the device, as shown in Fig. 2.
  • gas inlet 20 At the same time, it is necessary to supply an appropriate atmosphere, and the required gases can be charged through gas inlet 20.
  • the gas introduced through this inlet into the upper chamber 28 flows through the upper gas conduit 46A down to the middle chamber 30. From there, the gas also flows to the lower chamber 32 through the lower gas conduit 46B. In this way, gas is supplied evenly to all three chambers.
  • the upper gas conduit 46A have its top opening at a higher level than the top opening of the upper liquid conduits 42. This arrangement causes liquid to overflow once it reaches the top of the upper liquid conduit 42, and prevents the liquid from reaching the top of the upper gas conduit 46A, which could interfere with distribution of gas to the other chambers.
  • the lower gas conduit 46B has its top opening at a higher level than the top opening of the lower liquid conduits 44. Also, the bottom opening of the upper gas conduit 46A is located at or just below the upper divider member 24, so it can discharge gas into a region of the middle chamber 30 that will always be above the liquid level in that chamber. Likewise, the bottom opening of the lower gas conduit 46B is located at or just below the lower divider member 26.
  • FIG. 6 Another specific embodiment of the present invention is shown in Fig. 6.
  • This embodiment has eight cell culture devices 50A-50H stacked one on top of another. This arrangement permits a much greater quantity of cells to be cultured without taking up an excessive amount of floor space.
  • Each of the devices 50A-50H has three internal chambers and fluid communication means between those chambers as described above. For the sake of simplicity, those internal features are not shown in Fig. 6.
  • liquid inlets 52A-52H, the gas inlets 54A-54H, and the liquid outlets 56A-56H are all located on the same side of the devices 50A-50H.
  • Each liquid inlet 52 is connected by a liquid feed conduit, such as 58A and 58B, to a supply of the liquid media needed for cell culture, such as the bottles 60A and 60B.
  • Gravity, or some pumping means, can be used to cause a steady flow of the bottles' contents through the liquid feed conduits 58 into each cell culture device 50.
  • the gas inlets 54 are connected by gas feed conduits, such as 62A and 62B, to a supply of the gas needed for cell culture, such as pressure vessel 64. This arrangement allows the gas to be supplied as needed to cell culture devices 50 through the gas feed conduits 62. Valves, such as 66A and 66B, may be used to regulate or stop the gas flow.
  • the liquid outlets 56 are connected by liquid drain conduits, such as 68A and 68B, to a waste receptacle 70. This receptacle receives liquid that has flowed through the devices 50.
  • All the devices, including 50C-50H, would be connected by conduits as described above to supplies of liquid, supplies of gas, and waste receptacles. However, for the sake of simplicity, only the conduits for the devices 50A and 5OB are shown in Fig 5.
  • each of the eight stacked cell culture devices is 30 cm wide and long and 4.5 cm tall.
  • Fig. 5 illustrates how this would be divided among the three chambers.
  • the device 80 has an upper plate 82, a bottom plate
  • liquid inlet 88 gas inlet 90, and liquid outlet 92, which are connected to a liquid feed conduit 94, a gas feed conduit 96, and a liquid drain conduit 98, respectively.
  • the upper chamber has a gas region 100A and a liquid region 100B.
  • the middle chamber and lower chamber have similar gas and liquid regions 102A and 102B, and 104A and 104B, respectively.
  • the height of the liquid regions 100B, 102B, and 104B is defined by the height of the upper liquid conduits 106 (only one of which is shown) , the lower liquid conduits 108 (only one of which is shown) , and the liquid outlet 92. Once the liquid level reaches the top of either of those conduits, it overflows into the chamber below, or, in the case of the lower chamber, drains out through the liquid outlet 92.
  • the upper gas conduit 110 and lower gas conduit 112 permit the gas supplied through the gas inlet 90 to be distributed evenly to all three chambers.
  • Each gas conduit 110 and 112 is taller than the corresponding liquid conduit 106 and 108, and has its bottom opening at an altitude higher than that of the bottom opening of the corresponding liquid conduit 106 and 108, so that liquid will not flow through the gas conduits 110 and 112. If the thickness of the upper plate 82, bottom plate 84, upper divider member 114, and lower divider member 116 are each about 2 mm, then the liquid regions 100B, 102B, and 104B can suitably have a height of approximately 10 mm each, and the gas regions can suitably have a height of approximately 3 mm each. If each of the horizontal dimensions of the device were 30 cm, then the total volume of liquid in the device at any given time would be approximately 27 liters.
  • the remaining cells were inoculated into 0.8 1 culture flasks containing 100 ml of a growth medium which consisted of Iscove's medium (Gibco) supplemented with 10% of preselected fetal calf serum (Flow) , 10% of a preselected horse serum (Flow) , and hydrocortisone hemisuccinate at a final concentration of 5 x 10 ⁇ 7 mol/1. This gave a final cell concentration of about 2 x 10 6 cells/ml.
  • the culture medium contained 1.5 U/ml of preservative-free heparin.
  • the cultures were then gassed with air plus 5% C0 2 and incubated at 33 * C. Sixty culture flasks were established in this way.
  • the contents of the flasks including the adherent cells which were removed with a "rubber policeman," were decanted into plastic transfer packs (Travenol R1359) .
  • the bags were then centrifuged at 800 g for 20 minutes at 4"C.
  • the cell-free supernatant was removed with a Fenwal cell packer and the cells were then pooled and resuspended in equal volumes of 4.5% human albumin.
  • a total of 6.7 x 10 9 nucleated cells (equivalent to 1.35 x 10 8 cells/kg bodyweight) were collected and these processed marrow cells were transfused into the patient.
  • the present invention can be used with basically this same procedure, except that cell culture devices in accordance with the present invention should be substituted for the flasks described by Chang.
  • Additional growth medium is supplied on a continuous basis from supply bottles, such as 60A and 60B.
  • supply bottles such as 60A and 60B.
  • These bottles can be standard drip bottles which operate by gravity, or can be some other type of reservoir with suitable pumping means.
  • a specific procedure for using the present invention is as follows.
  • the system which has eight stacked devices as shown in Fig. 6 with a total liquid volume of 27 1, the system can be filled with liquid (cells plus growth medium) over a period of about 30 minutes.
  • the system should be allowed to settle for about 24-48 hours, and then a slow drip of liquid should be started at a total rate of about 0.27 1/day, or about 0.19 cc/min, divided evenly between the eight devices. This will result ideally in total replacement of the 27 1 of medium in about 10 days. Liquid is of course drained off at the same rate.
  • Incubation should be allowed to continue for about 7- 12 days, preferably about 10 days. During this time, the liquid supply bottles can be replaced as necessary. At the end of the inc ⁇ ibation time, the system is trypsinized and the liquid contents are collected by draining them through either the liquid drain conduit, or the liquid feed conduit (after inverting the device) .

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Abstract

A semi-closed cell culture device is disclosed which has three separate chambers (28, 30, 32), each divided from the others by horizontal plates (24, 26). Cells and liquid media are introduced into the uppermost chamber (28), from which liquid conduits carry them to the other two chambers (30, 32). Means are provided to supply the necessary atmosphere to each chamber. A continuous flow of liquid media can be supplied to the device, with an equal volume being constantly drained from the device, thereby eliminating the need to manually replace the media during the cell culture operation.

Description

DYNAMIC FLOW CELL CULTURE SYSTEM
The present invention relates to devices for culturing cells.
There are a number of situations in which it is useful to culture cells. One such situation is autologous bone marrow transplantation. In that procedure, bone marrow is harvested from a leukemia patient and is cultured in vitro under conditions that favor the growth of normal cells but cause leukemic cells to die out. The end result is a culture which should be substantially free of cancerous cells. The normal cells are recovered and transplanted back into the patient.
One serious drawback to such a procedure involves the devices in which the cell culture is done. Typically, 1 liter bottles are used, and often dozens of them are needed to handle the desired volume. Such a large number of bottles occupies a great deal of floor space in relation to the volume they hold. Further, the liquid medium in the bottles usually has to be changed every 3-5 days. The changing of the medium introduces a source of potential contamination, as well as requiring a significant amount of time and effort.
A long-standing need exists for improved cell culture devices which can perform the desired function without requiring so much space and attention and without such a large risk of contamination. The present invention concerns a cell culture device which includes a bottom plate; an upper enclosing member; a side enclosure which, together with the bottom plate and upper enclosing member, defines a completely enclosed volume; at least one horizontal divider member which is located between the bottom plate and the upper enclosing member and is attached around its perimeter to the side enclosure, thereby dividing the enclosed volume into at least two chambers, one of which is an upper chamber and one of which is a lower chamber; at least one liquid conduit traversing each horizontal divider member, the liquid conduit having a top opening and a bottom opening, with the liquid conduit extending upward above the horizontal divider member so that its top opening is located in the chamber above the horizontal divider member at a selected height, with the liquid conduit also extending downward below the horizontal member so that its bottom opening is located in the chamber below the horizontal divider member at a selected height; at least one gas conduit traversing each horizontal divider member, the gas conduit having a top opening and a bottom opening, with the gas conduit extending upward above the horizontal divider member so that its top opening is located in the chamber above the horizontal divider member at a level above that of the top opening of the liquid conduit in that chamber, with the gas conduit also extending downward below the horizontal divider member so that its bottom opening is located in the chamber below the horizontal divider member at a level above that of the bottom opening of the liquid conduit in that chamber; and inlet and outlet openings in the device for introducing and removing liquid and gas.
The bottom plate, upper enclosing member, and each horizontal divider are preferably flat plates which are parallel to each other. The side enclosure preferably is a number of flat vertical plates which are perpendicular to the bottom plate. The device can also include at least one vertical support post which extends between the upper enclosing member and a horizontal divider member, between a horizontal divider member and the bottom plate, or between two horizontal divider members. The inlet and outlet openings preferably include a liquid inlet and a gas inlet, both of which connect with the upper chamber, and a liquid outlet, which connects with the lower chamber.
In a particular embodiment, a cell culture device in accordance with the present invention includes a flat bottom plate; a flat upper plate; four rectangular side walls which are joined to each other and to the bottom plate and upper plate, thereby defining a completely enclosed volume; an upper divider member which is a flat plate parallel to the bottom plate and the upper plate and located inbetween them, and which is joined around its perimeter to the side walls; a lower divider member which is a flat plate parallel to the bottom plate and the upper plate and located inbetween the bottom plate and the upper divider member, and which is joined around its perimeter to the side walls, whereby the enclosed volume is divided into an upper chamber, a middle chamber, and a lower chamber, each of which has approximately equal volume; at least one upper liquid conduit traversing the upper divider member, the upper liquid conduit having a top opening and a bottom opening, with the upper liquid conduit extending upward above the upper divider member so that the top opening is located in the upper chamber at a selected height, the upper liquid conduit also extending downward below the upper divider member so that its bottom opening is located in the middle chamber at a selected height; at least one lower liquid conduit traversing the lower divider member, the lower liquid conduit having a top opening and a bottom opening, with the lower liquid conduit extending upward above the lower divider member so that the top opening is located in the middle chamber at a selected height, with the lower liquid conduit also extending downward below the lower divider member so that its bottom opening is located in the lower chamber at a selected height; at least one upper gas conduit traversing the upper divider member, the upper gas conduit having a top opening and a bottom opening, with the upper gas conduit extending upward above the upper divider member so that its top opening is located in the upper chamber at a level above that of the top opening of the upper liquid conduit, with the upper gas conduit also extending downward below the upper divider member so that its bottom opening is located in the middle chamber at a level above that of the bottom opening of the upper liquid conduit; at least one lower gas conduit traversing the lower divider member, the lower gas conduit having a top opening and a bottom opening, with the lower gas conduit extending upward above the lower divider member so that its top opening is located in the middle chamber at a level above that of the top opening of the lower liquid conduit, with the lower gas conduit also extending downward below the lower divider member so that its bottom opening is located in the lower chamber at a level above that of the bottom opening of the lower liquid conduit; and inlet and outlet openings in the device for introducing and removing liquid and gas.
The present invention also relates to a dynamic flow cell culture system, which includes a cell culture device as described above; a liquid feed conduit which connects at one end to the cell culture device and connects at the other end to a supply of liquid needed for cell culture, and supplies a flow of the liquid to the device; a gas feed conduit which connects at one end with the cell culture device and connects at the other end to a supply of gas needed for cell culture, and supplies a flow of the gas as needed to the device; and a liquid drain conduit which connects at one end with the cell culture device, and drains a flow of liquid from the device. In a preferred embodiment, at least one additional cell culture device as described above is stacked on top of another one, allowing the overall volume to be increased without taking up additional floor space.
In a dynamic flow cell culture system in accordance with the present invention, cells to be cultured are introduced into a sterilized cell culture device along with a suitable liquid growth medium. The device is then supplied with an atmosphere which consists essentially of air plus approximately 5% C02, and the cells are incubated. After a sufficient time has passed, the contents of the device can be removed, either through the liquid drain conduit, or through the liquid feed conduit after inverting the device.
The present invention provides a semi-closed system for cell culture which occupies less space and requires less effort than prior systems. Further, the present invention reduces the risk of contamination by eliminating the need for the cell culture device to be opened during the culturing process. The present invention is useful for purging tumor cells from the bone marrow of patients who have diseases such as chronic myeloid leukemia, acute myeloid leukemia, acute lymphocytic leukemia, myeloidplastic syndrome, and lymphoma. It is also useful for a variety of other cell culture operations.
Fig. 1 is a perspective cut-away view of a cell culture device in accordance with the present invention.
Fig. 2 is a top view of a cell culture device in accordance with the present invention. Fig. 3 and Fig. 4 are side cross sectional views of the device shown in Fig. 2, taken along the axes designated 3-3 and 4-4 in Fig. 2, respectively.
Fig. 5 shows a side cross sectional view of a cell culture device in accordance with the present invention while in operation.
Fig. 6 shows a dynamic flow cell culture system in accordance with the present invention.
The structure of a specific embodiment of a cell culture device in accordance with the present invention is shown in Figures 1-4. The device 10 has flat exterior surfaces with a rectangular cross-section. The device 10 has an upper plate 12, a bottom plate 14, and four side walls 16, which together define a completely enclosed space. The only openings in these surfaces and walls are a liquid inlet 18, a gas inlet 20, and a liquid outlet 22. The device 10 also has two internal horizontal dividers, which are an upper divider member 24 and a lower divider member 26. These two dividers, in conjunction with the upper plate 12 and bottom plate 14 and the side walls 16 define three separate enclosed chambers: an upper chamber 28, a middle chamber 30, and a lower chamber 32. These three chambers have fluid communication means that will be described below. The liquid inlet 18 and gas inlet 20 connect the upper chamber 28 with the outside of the device, while the liquid outlet 22 connects the lower chamber 32 with the outside.
Each of the upper plate 12, bottom plate 14, upper divider member 24, and lower divider member 26 are horizontal and are parallel to each other. Opposite side walls 16 are vertical and parallel to each other. Four support posts 40 are provided which effectively extend from the upper plate 12 to the bottom plate 14, and provide additional structural support for the device 10. These support posts 40 can be hollow cylinders which extend through circular holes cut in the upper divider member 24 and the lower divider member 26. However, it is preferred to construct each support post 40 out of three separate cylindrical members 40A, 40B, and 40C. The cylindrical member 40A extends vertically between the upper plate 12 and the upper divider member 24. The other two cylindrical members 40B and 40C extend in the same way between the upper divider member 24, the lower divider member 26, and the bottom plate 14.
The top view of the device 10 shown in Fig. 2 illustrates the position of the fluid communication means between the three chambers 28, 30, and 32.
The four support posts 40 are evenly spaced in order to provide the maximum structural support. There are also four upper liquid conduits 42 which connect the upper chamber 28 and the middle chamber 30. Likewise, there are four lower liquid conduits 44 which connect the middle chamber 30 and the lower chamber 32. In addition, in the center of the device 10 is a set of gas conduits 46.
The operation of these fluid communication means can be understood more easily by referring to Figs. 3 and 4. Fig. 4 is a side cross-sectional view of the device 10 taken along the axis designated 4-4 in Fig. 2. Looking from this side view one sees the support posts 40A, 40B, and 40C. On either side are lower liquid conduits 44 which connect the middle chamber 30 and the lower chamber 32. These conduits 44 preferably are hollow cylindrical tubes and are fixed in place in openings in the lower divider member 26. The openings in the member 26 should be no larger than necessary to insert the channel 44 and should have the same horizontal cross-sectional shape, so as to prevent fluid from flowing from one chamber to another chamber below it by seeping between the outside of the cylindrical tube and the divider member. Above and in the background can be seen upper liquid conduits 42.
Referring now to Fig. 3, which is a side cross- sectional view of the device 10 taken along the axis designated 3-3 in Fig. 2, one can again see a lower liquid conduit 44 which connects the middle chamber 30 and the lower chamber 32. One also sees two upper liquid conduits 42 which connect the upper chamber 28 and the middle chamber 30, in the same manner as described above for the lower conduits 44.
Referring back to Fig. 4, the set of gas conduits includes an upper gas conduit 46A and a lower gas conduit 46B. The upper gas conduit 46A connects the upper chamber 28 and the middle chamber 30, while the lower gas conduit 46B connects the middle chamber 30 and the lower chamber 32.
Each component of the device, including the upper plate 12, bottom plate 14, side walls 16, upper divider member 24, lower divider member 26, support posts 40, liquid conduits 42 and 44, and gas conduits 46A and 46B, are preferably transparent. Suitable materials include plastic and glass.
Operation begins by sterilizing the device 10, and then charging liquid, such as bone marrow in appropriate liquid media, through the liquid inlet 18. The liquid spills into the upper chamber 28, .and the liquid level in that chamber rises as more liquid is charged. When the liquid level reaches the top of the upper liquid conduits 42, it begins to spill through those conduits into the middle chamber 30. As still more liquid is charged through the liquid inlet 18, the liquid level rises in the middle chamber 30 until it reaches the top of the lower liquid conduits 44. At that point, the liquid begins to spill through the lower liquid conduits 44 into the lower chamber 32. In this manner, liquid can be evenly distributed to all three chambers. Evenness of distribution is enhanced by locating the liquid conduits 42 and 44 in a regular pattern across the device, as shown in Fig. 2.
At the same time, it is necessary to supply an appropriate atmosphere, and the required gases can be charged through gas inlet 20. The gas introduced through this inlet into the upper chamber 28 flows through the upper gas conduit 46A down to the middle chamber 30. From there, the gas also flows to the lower chamber 32 through the lower gas conduit 46B. In this way, gas is supplied evenly to all three chambers.
It is important that the upper gas conduit 46A have its top opening at a higher level than the top opening of the upper liquid conduits 42. This arrangement causes liquid to overflow once it reaches the top of the upper liquid conduit 42, and prevents the liquid from reaching the top of the upper gas conduit 46A, which could interfere with distribution of gas to the other chambers. In the same way, the lower gas conduit 46B has its top opening at a higher level than the top opening of the lower liquid conduits 44. Also, the bottom opening of the upper gas conduit 46A is located at or just below the upper divider member 24, so it can discharge gas into a region of the middle chamber 30 that will always be above the liquid level in that chamber. Likewise, the bottom opening of the lower gas conduit 46B is located at or just below the lower divider member 26.
Another specific embodiment of the present invention is shown in Fig. 6. This embodiment has eight cell culture devices 50A-50H stacked one on top of another. This arrangement permits a much greater quantity of cells to be cultured without taking up an excessive amount of floor space. Each of the devices 50A-50H has three internal chambers and fluid communication means between those chambers as described above. For the sake of simplicity, those internal features are not shown in Fig. 6.
In this embodiment, the liquid inlets 52A-52H, the gas inlets 54A-54H, and the liquid outlets 56A-56H are all located on the same side of the devices 50A-50H. Each liquid inlet 52 is connected by a liquid feed conduit, such as 58A and 58B, to a supply of the liquid media needed for cell culture, such as the bottles 60A and 60B. Gravity, or some pumping means, can be used to cause a steady flow of the bottles' contents through the liquid feed conduits 58 into each cell culture device 50.
The gas inlets 54 are connected by gas feed conduits, such as 62A and 62B, to a supply of the gas needed for cell culture, such as pressure vessel 64. This arrangement allows the gas to be supplied as needed to cell culture devices 50 through the gas feed conduits 62. Valves, such as 66A and 66B, may be used to regulate or stop the gas flow.
The liquid outlets 56 are connected by liquid drain conduits, such as 68A and 68B, to a waste receptacle 70. This receptacle receives liquid that has flowed through the devices 50.
All the devices, including 50C-50H, would be connected by conduits as described above to supplies of liquid, supplies of gas, and waste receptacles. However, for the sake of simplicity, only the conduits for the devices 50A and 5OB are shown in Fig 5.
The dimensions of the components can be varied, but in one embodiment, each of the eight stacked cell culture devices is 30 cm wide and long and 4.5 cm tall. Fig. 5 illustrates how this would be divided among the three chambers.
The device 80 has an upper plate 82, a bottom plate
84, and side walls 86. It also has a liquid inlet 88, gas inlet 90, and liquid outlet 92, which are connected to a liquid feed conduit 94, a gas feed conduit 96, and a liquid drain conduit 98, respectively.
The upper chamber has a gas region 100A and a liquid region 100B. The middle chamber and lower chamber have similar gas and liquid regions 102A and 102B, and 104A and 104B, respectively. The height of the liquid regions 100B, 102B, and 104B is defined by the height of the upper liquid conduits 106 (only one of which is shown) , the lower liquid conduits 108 (only one of which is shown) , and the liquid outlet 92. Once the liquid level reaches the top of either of those conduits, it overflows into the chamber below, or, in the case of the lower chamber, drains out through the liquid outlet 92. The upper gas conduit 110 and lower gas conduit 112 permit the gas supplied through the gas inlet 90 to be distributed evenly to all three chambers. Each gas conduit 110 and 112 is taller than the corresponding liquid conduit 106 and 108, and has its bottom opening at an altitude higher than that of the bottom opening of the corresponding liquid conduit 106 and 108, so that liquid will not flow through the gas conduits 110 and 112. If the thickness of the upper plate 82, bottom plate 84, upper divider member 114, and lower divider member 116 are each about 2 mm, then the liquid regions 100B, 102B, and 104B can suitably have a height of approximately 10 mm each, and the gas regions can suitably have a height of approximately 3 mm each. If each of the horizontal dimensions of the device were 30 cm, then the total volume of liquid in the device at any given time would be approximately 27 liters.
The presently preferred procedure for cell culture using the present invention is described by Chang et al, "Reconstitution of Haemopoietic System With Autologous Marrow Taken During Relapse of Acute Myeloblastic Leukaemia and Grown in Long-Term Culture," Lancet, 294-295, Feb. 8, 1986, which is incorporated here by reference. That article describes a procedure in which 1.7 liters of marrow was harvested, and approximately 21 x 109 nucleated cells were collected in heparinised (10 U/ml) containers. Excess red blood cells were removed by centrifugation and 8 x 109 cells were frozen in liquid N2 as reserve. The remaining cells were inoculated into 0.8 1 culture flasks containing 100 ml of a growth medium which consisted of Iscove's medium (Gibco) supplemented with 10% of preselected fetal calf serum (Flow) , 10% of a preselected horse serum (Flow) , and hydrocortisone hemisuccinate at a final concentration of 5 x 10~7 mol/1. This gave a final cell concentration of about 2 x 106 cells/ml.
To prevent coagulation, the culture medium contained 1.5 U/ml of preservative-free heparin. The cultures were then gassed with air plus 5% C02 and incubated at 33* C. Sixty culture flasks were established in this way.
After 10 days of culture, the contents of the flasks, including the adherent cells which were removed with a "rubber policeman," were decanted into plastic transfer packs (Travenol R1359) . The bags were then centrifuged at 800 g for 20 minutes at 4"C. The cell-free supernatant was removed with a Fenwal cell packer and the cells were then pooled and resuspended in equal volumes of 4.5% human albumin. By this procedure, a total of 6.7 x 109 nucleated cells (equivalent to 1.35 x 108 cells/kg bodyweight) were collected and these processed marrow cells were transfused into the patient.
The present invention can be used with basically this same procedure, except that cell culture devices in accordance with the present invention should be substituted for the flasks described by Chang.
Additional growth medium is supplied on a continuous basis from supply bottles, such as 60A and 60B. These bottles can be standard drip bottles which operate by gravity, or can be some other type of reservoir with suitable pumping means.
A specific procedure for using the present invention is as follows. In a system which has eight stacked devices as shown in Fig. 6 with a total liquid volume of 27 1, the system can be filled with liquid (cells plus growth medium) over a period of about 30 minutes. The system should be allowed to settle for about 24-48 hours, and then a slow drip of liquid should be started at a total rate of about 0.27 1/day, or about 0.19 cc/min, divided evenly between the eight devices. This will result ideally in total replacement of the 27 1 of medium in about 10 days. Liquid is of course drained off at the same rate.
Incubation should be allowed to continue for about 7- 12 days, preferably about 10 days. During this time, the liquid supply bottles can be replaced as necessary. At the end of the incτibation time, the system is trypsinized and the liquid contents are collected by draining them through either the liquid drain conduit, or the liquid feed conduit (after inverting the device) .
The preceding description is intended to describe various specific embodiments of the present invention. It is not intended to provide an exhaustive description of all possible embodiments of the present invention. Those skilled in this field will recognize that modifications could be made to the specific embodiments that are disclosed which would remain within the scope of the present invention.

Claims

_____[____'■
1. A cell culture device, including:
a bottom plate;
an upper enclosing member;
a side enclosure which, together with the bottom plate and upper enclosing member, defines a completely enclosed volume;
at least one horizontal divider member which is located between the bottom plate and upper enclosing member and is attached around its perimeter to the side enclosure, thereby dividing the enclosed volume into at least two chambers, one of which is an upper chamber and one of which is a lower chamber;
at least one liquid conduit traversing each horizontal divider member, the liquid conduit having a top opening and a bottom opening, with the liquid conduit extending upward above the horizontal divider member so that its top opening is located in the chamber above the horizontal divider member at a selected height, with the liquid conduit also extending downward below the horizontal member so that its bottom opening is located in the chamber below the horizontal divider member at a selected height;
at least one gas conduit traversing each horizontal divider member, the gas conduit having a top opening and a bottom opening, with the gas conduit extending upward above the horizontal divider member so that its top opening is located in the chamber above the horizontal divider member at a level above that of the top opening of the liquid conduit in that chamber, with the gas conduit also extending downward below the horizontal divider member so that its bottom opening is located in the chamber below the horizontal divider member at a level above that of the bottom opening of the liquid conduit in that chamber; and
inlet and outlet openings in the device for introducing and removing liquid and gas.
2. The device of claim 1, where there are two horizontal divider members, dividing the enclosed volume into an upper chamber, middle chamber, and lower chamber.
3. The device of claim 1, where the bottom plate, upper enclosing member, and each horizontal divider are flat plates which are parallel to each other.
4. The device of claim 1, where the side enclosure consists of four flat vertical plates.
5. The device of claim 1, further including vertical support posts extending between the upper enclosing member, each horizontal divider member, and the bottom plate.
6. The device of claim one where the inlet and outlet openings include a liquid inlet and a gas inlet, both of which connect with the upper chamber, and a liquid outlet, which connects with the lower chamber.
7. A cell culture device, including:
a flat bottom plate;
a flat upper plate;
four rectangular side walls which are joined to each other and to the bottom plate and upper plate, thereby defining a completely enclosed volume;
an upper divider member which is a flat plate parallel to the bottom plate and the upper plate and located inbetween them, and which is joined around its perimeter to the side walls;
a lower divider member which is a flat plate parallel to the bottom plate and the upper plate and located inbetween the bottom plate and the upper divider member, and which is joined around its perimeter to the side walls, whereby the enclosed volume is divided into an upper chamber, a middle chamber, and a lower chamber, each of which has approximately equal volume;
at least one upper liquid conduit traversing the upper divider member, the upper liquid conduit having a top opening and a bottom opening, with the upper liquid conduit extending upward above the upper divider member so that the top opening is located in the upper chamber at a selected height, the upper liquid conduit also extending downward below the upper divider member so that its bottom opening is located in the middle chamber at a selected height;
at least one lower liquid conduit traversing the lower divider member, the lower liquid conduit having a top opening and a bottom opening, with the lower liquid conduit extending upward above the lower divider member so that its top opening is located in the middle chamber at a selected height, with the lower liquid conduit also extending downward below the lower divider member so that its bottom opening is located in the lower chamber at a selected height;
at least one upper gas conduit traversing the upper divider member, the upper gas conduit having a top opening and a bottom opening, with the upper gas conduit extending upward above the upper divider member so that its top opening is located in the upper chamber at a level above that of the top opening of the upper liquid conduit, with the upper gas conduit also extending downward below the upper divider member so that its bottom opening is located in the middle chamber at a level above that of the bottom opening of the upper liquid conduit;
at least one lower gas conduit traversing the lower divider member, the lower gas conduit having a top opening and a bottom opening, with the lower gas conduit extending upward above the lower divider member so that its top opening is located in the middle chamber at a level above that of the top opening of the lower liquid conduit, with the lower gas conduit also extending downward below the lower divider member so that its bottom opening is located in the lower chamber at a level above that of the bottom opening of the lower liquid conduit; and
inlet and outlet openings in the device for introducing and removing liquid and gas.
8. The device of claim 7, here there are four upper liquid conduits and four lower liquid conduits.
9. The device of claim 7, further including at least one support post extending between each of the upper plate and the upper divider member, the upper divider member and the lower divider member, and the lower divider member and the bottom plate.
10. The device of claim 7, further including four support posts extending between each of the upper plate and the upper divider member, the upper divider member and the lower divider member, and the lower divider member and the bottom plate.
11. The device of claim 7, where the side walls are perpendicular to the upper plate, upper divider member, lower divider member, and bottom plate.
12. The device of claim 7, where inlet and outlet openings consist of a liquid inlet which connects with the upper chamber, a gas inlet which connects with the upper chamber, and a liquid outlet which connects with the lower chamber.
13. A dynamic flow cell culture system, including:
a cell culture device in accordance with claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12;
a liquid feed conduit which connects at one end to the cell culture device and connects at the other end to a supply of liquid needed for cell culture, and supplies a flow of the liquid to the device;
a gas feed conduit which connects at one end with the cell culture device and connects at the other end to a supply of gas needed for cell culture, and supplies a flow of the gas as needed to the device; and
a liquid drain conduit which connects at one end with the cell culture device, and drains a flow of liquid from the device.
14. The system of claim 13, further including at least one additional cell culture device in accordance with claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, where the cell culture devices are stacked one on top of another, and further including an additional liquid feed conduit, gas feed conduit, and liquid drain conduit for each additional cell culture device.
PCT/US1990/000480 1989-06-14 1990-01-24 Dynamic flow cell culture system WO1990015859A1 (en)

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WO2017066821A1 (en) * 2015-10-19 2017-04-27 Skin Tissue Engineering Pty Ltd Apparatus and products for culturing of cells or tissues

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