WO2012096559A1

WO2012096559A1 - A cell culture tank

Info

Publication number: WO2012096559A1
Application number: PCT/MY2011/000190
Authority: WO
Inventors: Lai Keng CHAN; Peng Lim BOEY
Original assignee: Universiti Sains Malaysia
Priority date: 2011-01-14
Filing date: 2011-08-18
Publication date: 2012-07-19
Also published as: JP2014502512A; MY148876A; KR20140003539A

Abstract

A cell culture apparatus (100) comprising a culture tank (110) for growing plant cell in a culture medium, having an upper portion (121) and a lower portion (122); a sterilizing means (107) encompassing the upper portion (121) of the culture tank (110) for sterilizing the culture tank (110); a cooling system (109) enclosing the lower portion (122) of the culture tank for stabilizing the temperature; a lighting means (106) fitted within the upper portion (121) of the culture tank (110) for providing the necessary light intensity and light colour to the cell culture; an aerating means (123) fitted within the lower portion (122) of the culture tank (110) for supplying air to the cell culture; and a covering means (120) having a plurality of openings for insertion of various applicators for providing predetermined growth conditions to the cell culture.

Description

A CELL CULTURE TANK

FIELD OF INVENTION

The present invention relates to a culturing or cultivating apparatus for plant cells. More particularly, the present invention relates to a plant cell culture tank which is innovatively incorporated with an efficient sterilization system, a cooling system and a multi-factor-controlling system, and suitable for use in mass-producing any plant cells.

BACKGROUND OF THE INVENTION Cell culture is a process for growing cells under controlled conditions. The are various types of techniques used in the existing technology for culturing different types of cells. The techniques for plant cell culture are usually different from that of the animal cell culture. Factors affecting cell growth for each plant species are different and unique for each particular species. Optimization for optimum cell growth hence need to be carried out for each plant species.

Plant cell culture is a practice increasingly used by the researcher in the field of plant science to propagate plant cells or tissues for cloning and especially for production of useful secondary metabolites. Such a practice is significantly useful, especially in various biotechnological and pharmaceutical applications. It can be used in the production of secondary metabolites, development of herbicides, production of plant hormones and other commercial applications. Therefore, mass production of a particular plant cell by an effective cell culture technique is necessary. A suitable and efficient cell culture system become crucial and important factor for the successful and sustainable production of the end products. Conventional plant cell culture apparatus, such as the stirred-tank reactor, may not be suitable for mass-cultivation of the plant cells. Hence, an innovative cell culture tank is vital to ensure the productivity and quality of the plant cells produced. As the process of cultunng plant cell culture has to be conducted under certain controlled conditions, a cell culture tank which is incorporated with all the essential systems or components is desirable in order to achieve the desired conditions for the growth of the plant cells. These essential systems shall include a sterilization, and a multi-factor- controlling system, such as a temperature-pH-aeration-illumination-controlling system.

There are a few existing technologies disclosed in the prior art relating to cell culturing or cultivating apparatus. Most of the apparatus is an incubator or involving an incubator unit. Disclosed in U.S. Patent No. 2006151185 is an automatic cell cultivation apparatus utilizing autoclave sterilization and method for using the same. This automatic cell cultivation apparatus comprises an operation unit for operating to cultivate cells of subjects, incubator units for cultivating the cells, a cold storage unit for storing reagent, a normal temperature storage unit for storing culture tools, and an inlet-outlet unit for taking in and out reagents and culture tools. A steam supply unit for autoclave sterilization is provided, in which the incubator units, the cold storage unit, the normal temperature storage unit and the inlet-outlet unit communicate with the operation unit. This cell cultivation apparatus involves various components, thus it is complicated and not portable. It does not relate to a culture tank which is suitable to be used for culturing any types of plant cells. Moreover, it requires an autoclave sterilization system which is not economical.

Another U.S. Patent No. 4696902 discloses a modular apparatus for cell culture comprising an enclosure with isoterm walls, at least one device for storing culture containers, at least one temperature regulation device, a closed circuit filtered laminar flow sterilization device, a device for supplying and regulating gas, such as air, carbon dioxide and vapour. There is also at least one additional device for carrying out any one of the conventional cell culture operations or any other specific operation provided in the enclosure, and defining the modular work post chosen depending on the needs. This apparatus may also comprise an automatic and programmable handling arm and a microprocessor for controlling and processing the data. Even though a sterilization and a temperature regulation device are provided, this apparatus, however, does not involve a culture tank which can mass-produce the plant cells.

A Japan Patent No. 4020280 also discloses a cultivation apparatus, which is a gaseous phase cultivation apparatus. A thermal sterilization and temperature-controlling means is placed outside a cultivation tank to enable the thermal sterilization and the temperature control in the tank. An implantation hole for charging a cultivation object and an aeration hole are formed at the upper part of the cultivation tank and the bottom of the tank is provided with a cultivation liquid holding part. However, this apparatus cultivates the cell in gaseous phase and not in liquid phase. It is also not an integrated system, not portable and may not be suitable for culturing any types of plant cells because it does not possess a flexible system.

In short, there is no existing technology in the prior art disclosing an integrated plant cell culture tank which has a sterilization, a cooling system and a multi-factor- controlling system, and can be fitted with various probes and equipments to provide the desired conditions for the growth of the cultured cells. Besides, the cell culture apparatus disclosed in the prior art are not portable. The construction and the building materials used for the cell culture tank also does not allow the observation of the production status and growth of the cultured cells. Clearly, there is a need for the present invention to provide an integrated plant cell culture tank to overcome the drawbacks of the prior art.

SUMMARY OF INVENTION The primary object of the present invention is to provide an integrated culture tank which is incorporated with various improved equipments and systems, suitable for cultivating and mass-producing various types of plant cells.

Another object of the present invention is to provide a flexible and versatile plant cell culture tank, in which various probes for determination of any desired parameters can be applied to provide the desired conditions for the cell culture process. Still another object of the present invention is to provide a plant cell culture tank having various functional features capable of improving the productivity of the cell culture.

Yet another object of the present invention is to provide a plant cell culture tank in which the production status and growth of the cultured cells can be easily observed by viewing, or can be monitored by a computer.

Further object of the present invention is to provide a plant cell culture tank with a self autoclaving system which is capable of preventing contamination of the cultured cells.

Another further object of the present invention is to provide a plant cell culture tank which is durable, portable, economical and commercially sustainable. At least one of the preceding objects is met, in whole or in part, by the present invention, in which one of the embodiments of the present invention describes a cell culture apparatus (100) comprising a culture tank (110) for growing plant cell in a culture medium, having an upper portion (121) and a lower portion (122); a sterilizing means (107) encompassing the upper portion (121) of the culture tank (110) for sterilizing the culture tank (110); a cooling system (109) enclosing the lower portion (122) of the culture tank for stabilizing the temperature; a lighting means (106) fitted within the upper portion (121) of the culture tank (110) for providing the necessary light intensity and light colour to the cell culture; an aerating means (123) fitted within the lower portion (122) of the culture tank (110) for supplying air to the cell culture; and a covering means (120) having a plurality of openings for insertion of various applicators for providing predetermined growth conditions to the cell culture.

One of the preferred embodiments of the present invention discloses that the culture tank (110) of the cell culture apparatus (100) is made of stainless steel.

Another preferred embodiment of the present invention discloses that the sterilizing means (107) is a band heater.

In accordance with yet another preferred embodiment of the present invention, the cooling system (109) can be a water jacket which has a water inlet (116) and a water outlet (108).

Still another preferred embodiment of the present invention discloses that the lighting means (106) is mounted on the covering means (120) and extended via an opening on the covering means (120) into the culture tank (110).

It is also disclosed in another preferred embodiment of the present invention that the aerating means (123) is connected to a pumping means (111) and an outputting means (112).

According to still another preferred embodiment of the present invention, the plurality of probes include a temperature sensor (101), a pH sensor (102), a dissolved oxygen (DO) sensor (103), an acid or base applicator (115) and an air-ventilator (105).

Further embodiment of the present invention is a cell culture apparatus (100) according which further comprises a stirrer (117) mounted on the covering means (120) and extended via an opening at the centre into the culture tank (110) for continuously stirring and mixing the cell culture in the culture tank (110). Preferably, there is a motor (104) connected to the stirrer (117) for its rotation.

Another further embodiment of the present invention is a cell culture apparatus (100) which further comprises a supporting means (124) onto which the culture tank (110) is fitted. According to another further embodiment of the present invention, the cell culture apparatus (100) further comprises a viewing window (125) on the culture tank (110) allowing a user to observe the conditions in the culture tank (110).

Preferably, the cell culture apparatus (100) is integrated with a computer program for monitoring and controlling the conditions in the culture tank (110). The conditions are preferably controlled via a control panel (200).

The plant cell culture apparatus (100) of the present invention serves as a bioreactor which is incorporated with an innovative sterilization and an effective cooling system within the same unit. It applies an efficient temperature-controlling system and eliminates the use of autoclave sterilization in which large autoclave instrument is involved. As it is not necessary for the culture tank (110) to be placed in the autoclave instrument, the size of the tank (110) will not be limited. Besides, the covering means (120) of the culture tank (110) can be fitted with various probes for determination of any desired parameters, and a viewing window (125) enabling one to observe the production status and growth of the cultured cells. Moreover, the culture tank (110) of the present invention is also durable, portable, economical and commercially sustainable. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The embodiments described herein are not intended as limitations on the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of facilitating an understanding of the invention, there is illustrated in the accompanying drawing the preferred embodiments from an inspection of which when considered in connection with the following description, the invention, its construction and operation and many of its advantages would be readily understood and appreciated.

Figure 1 is a cross-sectional view of the cell culture apparatus (100) as described in one of the preferred embodiments of the present invention.

Figure 2 is a top view of the covering means (120) or the cell culture apparatus

(100) of Figure 1. Figure 3 is a schematic diagram of the control panel (200) for the cell culture apparatus (100) of Figure 1.

Figure 4 is a front view of the cell culture apparatus (100) of Figure 1 showing the viewing window (125).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a culturing or cultivating apparatus for plant cells. More particularly, the present invention relates to a plant cell culture tank which is innovatively incorporated with an efficient sterilization system, a cooling system and a multi-factor-controlling system, and suitable for use in mass-producing any plant cells. Hereinafter, the invention shall be described according to the preferred embodiments of the present invention and by referring to the accompanying description and drawings. However, it is to be understood that limiting the description to the preferred embodiments of the invention and to the drawings is merely to facilitate discussion of the present invention and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim.

The present invention discloses a cell culture apparatus (100) comprising a culture tank (110) for growing plant cell in a culture medium, having an upper portion (121) and a lower portion (122); a sterilizing means (107) encompassing the upper portion (121) of the culture tank (110) for sterilizing the culture tank (110); a cooling system (109) enclosing the lower portion (122) of the culture tank for stabilizing the temperature; a lighting means (106) fitted within the upper portion (121) of the culture tank (110) for providing the necessary light intensity and light colour to the cell culture; an aerating means (123) fitted within the lower portion (122) of the culture tank (110) for supplying air to the cell culture; and a covering means (120) having a plurality of openings for insertion of various applicators for providing predetermined growth conditions to the cell culture.

Illustrated in Figure 1 is a cross-sectional view of the cell culture apparatus (100) as described in one of the preferred embodiments of the present invention, showing the overall layout of the apparatus (100). The cell culture apparatus (100) can serves as an integrated bioreactor suitable for culturing and cultivating various species of plant cells. This invented cell culture tanks have been proven to be suitable for mass production of plant cells and bioactive compounds from Tongkat Ali (Eurycoma longifolia Jack.), Misai Kucing (Orthosiphon stameneus), Rumput Ganda (Cyperus aromaticus) and Artemesia annua L. The type of cell culture medium used is depending on the species of the plant cells to be cultured.

In accordance with one of the preferred embodiments of the present invention, the culture tank (110) of the cell culture apparatus (100) is made of stainless steel. However, the present invention does not intend to limit the material used for the culture tank (110) provided that the material used is highly durable and not easily broken. This is different from the conventional type of culture tank or bioreator, which is usually made from glass material.

According to the preferred embodiment of the present invention, the culture tank (110) of the present invention can be fabricated into different sizes and dimensions according to its applications. The preferred sizes of the culture tank (110) include the 10L and the 20L tank, in which the tank (110) can be fabricated in such a way to fit in culture medium of 75% by volume of the 10 litre or 20 litre culture tank (110), respectively. For example, a 10L culture tank can have a height of approximately 30.5 - 50 cm with an internal diameter of approximately 16 - 20.5 cm and an external diameter of approximately 21 - 26 cm. Whilst, the 20L culture tank can be fabricated to have a height of approximately 40.0 - 50.0 cm with an internal diameter of approximately 22.5 - 27.2 cm and an external diameter of approximately 28 - 32.6 cm. Accordingly, the culture tank (110) is preferably fabricated into a cylindrical shape. Other suitable shapes can also be applied according to its applications or the preferences of users. As shown in Figure 1, the culture tank (110) is divided into an upper portion (121) and a lower portion (122). There is a sterilizing means (107) located at the upper portion (121) of the culture tank (110). As an example, the upper portion (121) of the tank (110) can be of approximately 15.5 - 25.5 cm in height for the 10L tank, and 19.6 - 25.0 cm in height for the 20L tank. Preferably, this sterilizing means (107) encompasses the outer wall of the culture tank (110) to provide a sterilizing facility to the plant cell cultured in the culture tank (110). According to a preferred embodiment of the present invention, the sterilizing means (107) is a band heater. Band heater is used herein because it can provide self sterilization of the cell culture in the culture tank (110).

Located at the lower portion (122) of the culture tank (110) is a cooling system (109), as shown in Figure 1. In accordance with another preferred embodiment of the present invention, the cooling system (109) can be a water jacket. This water jacket preferably encloses the lower portion (122) of the culture tank (110) for stabilizing the temperature of the cell cultures in the culture tank (110). The water jacket has a water inlet (116) located at a relatively lower position, and a water outlet (108) located at a relatively higher position of the lower portion (122) of the culture tank (110) in order to maintain the water current within the water jacket, and thus providing the cooling effect to the culture tank (110).

Besides the sterilizing means (107) and the cooling system (109), another important feature of the cell culture apparatus (100) as described in the preferred embodiment of the present invention is the lighting means (106). The lighting means is preferably mounted on the covering means (120) of the culture tank (110), and extended via an opening on the covering means (120) into the culture tank (110). There are many types of suitable lighting means (106) that can be applied in the present invention according to the required light intensity and light colour. The lighting means (106) used is preferably changed easily according to the necessary light intensity and the type of light colour required by the cell growth of a particular plant species, hence the cell culture apparatus (100) does not required any external light source for the optimum growth of the plant cells.

Besides, it is also disclosed in another preferred embodiment that the culture tank (110) of the present invention contains an aerating means (123), which is preferably having a three-way valve. The aerating means (123) is connected to a pumping means (111) and an outputting means (112), and preferably fitted at the lower portion (122) of the culture tank (110). The pumping means allows the external air to be pumped into the culture tank (110) to create aeration within the tank (110), whereas the outputting means (112) is used for collecting the output of the cultured cell products for sample-testing or harvesting purposes.

Figure 2 shows a top view of the covering means (120) or the cell culture apparatus (100) of Figure 1. According to the preferred embodiment of the present invention, the covering means (120) is preferably made of cast iron or other suitable materials with high tensile strength.

As shown in both Figure 1 and Figure 2, the covering means (120) contains a number of openings for the insertion of various applicators, each meant for providing respective predetermined growth conditions for the cell culture. According to still another preferred embodiment of the present invention, the applicators include a temperature sensor (101), a pH sensor (102), a DO sensor (103), an acid or base applicator (115) and an air-ventilator (105). Preferably, the temperature sensor (101) can be a thermocouple. The pH sensor (102) and the DO sensor (103) are respectively known as pH probe and DO probe. Whilst, the acid or base applicator (115) is used for regulating the pH of the culture medium and cell cultures in the tank (110). Besides, an exhaust air is used as the air- ventilator (105) for releasing the pressure formed within the culture tank (110). As set forth in the preceding description, there are also openings for the lighting means (106) located on the covering means (120). In addition, one or more spare openings (113, 114) can also be provided for the determination of other desired parameters for the optimum culture conditions in the culture tank (110). Therefore, the integrated cell culture apparatus (100) of the present invention is flexible and versatile.

Further embodiment of the present invention discloses that the integrated cell culture apparatus (100) further comprises a stirrer (117) mounted on the covering means (120) and extended via an opening at the centre of the covering means (120) into the culture tank (110) for continuously stirring and mixing the cells in the culture tank (110). Preferably, there is a motor (104) connected to the stirrer (117) for its rotation. The motorized stirrer (117) is preferably fitted at the centre of the covering means (120) to ensure the complete mixture of the culture medium and cultured cells within the tank (110).

Besides, the cell culture apparatus (100) also comprises a supporting means (124) onto which the culture tank (110) is fitted, according to another further embodiment of the present invention. The supporting means (124) is preferably a plurality of supporting legs. Similar to the covering means (120), the supporting means (124) of the culture tank (110) is made of cast iron or any other suitable material with high tensile strength and durable. According to another further embodiment of the present invention, the cell culture apparatus (100) further comprises a viewing window (125), which is preferably a view window, fitted on the culture tank (110) allowing a user to observe the cell growth conditions in the culture tank (110). Figure 4 shows the location of the viewing window (125) on the outer surface of the culture tank (110). As a result, the production status and growth of the cultured cells can be observed and monitored via this viewing window (125).

Preferably, the cell culture apparatus (100) is integrated with a computer program for monitoring and controlling the conditions in the culture tank (110). These conditions can be controlled via a control panel (200). As illustrated in Figure 3, the control panel (200) can contain various controlling buttons, including a pH and DO measurer (202), a temperature controlling button (201), a motorized stirrer controller (217), a speed controller (216), a timer (219), a light controller (206), an aeration controller (211), a heater controller (207) and a power switch (218). The pH button is used for measuring the pH of the cell culture and culture medium while the DO button is to measure the amount of dissolved oxygen to be added into the cell cultures or to measure the amount of dissolve oxygen contained in the cell culture. The temperature controlling button (201) controls the temperature of the inner tank during sterilization process and the temperature of the cell culture during growing process. The motorized stirrer controller (217) is used to rotate the stirrer (117). The speed controller (216) measures the speed of the stirrer (117). The timer (219) can measure the sterilization process duration and the cell culture duration; whereas the light controller (206) measures the light intensity. The aeration controller (211) measures the amount of air supply to the cell culture system, whereas the heater controller (207) measures the temperature of the heating band. The power switch is (218) provided to power on or off the electricity supply.

The cell culture apparatus (100) provided by the present invention is portable and easily transported, even by simple vehicles such as car or motorbike, hence it can be used anywhere as long as it is placed in a clean and temperature regulated room. The cell culturing system conducted by this apparatus (100) can be applied commercially with cheaper capital start-up, thus it is commercially sustainable. Since the apparatus (100) is made up of individual tank (110), contamination of the cell cultures therein can be easily controlled as compared to the conventionally available large bioreactor.

The present disclosure includes as contained in the appended claims, as well as that of the foregoing description. Although this invention has been described in its preferred form with a degree of particularity, it is understood that the present disclosure of the preferred form has been made only by way of example and that numerous changes in the details of construction and the combination and arrangements of parts may be resorted to without departing from the scope of the invention.

Claims

1. A cell culture apparatus ( 100) comprising:

a culture tank (110) for growing plant cell in a culture medium, having an upper portion (121) and a lower portion (122);

a sterilizing means (107) encompassing the upper portion (121) of the culture tank (110) for sterilizing the culture tank (110);

a cooling system (109) enclosing the lower portion (122) of the culture tank for stabilizing the temperature;

a lighting means (106) fitted within the upper portion (121) of the culture tank (110) for providing the necessary light intensity and light colour to the cell culture;

an aerating means (123) fitted within the lower portion (122) of the culture tank (110) for supplying air to the cell culture; and

a covering means (120) having a plurality of openings for insertion of various applicators for providing predetermined growth conditions to the cell culture.

2. A cell culture apparatus (100) according to claim 1, wherein the culture tank (110) is made of stainless steel.

3. A cell culture apparatus (100) according to claim 1, wherein the sterilizing means (107) is a band heater.

4. A cell culture apparatus (100) according to claim 1, wherein the cooling system (109) is a water jacket.

5. A cell culture apparatus (100) according to claim 1, wherein the lighting means (106) is mounted on the covering means (120) and extended via an opening on the covering means (120) into the culture tank (110).

6. A cell culture apparatus (100) according to claim 1, wherein the aerating means (123) is connected to a pumping means (111) and an outputting means (112).

7. A cell culture apparatus (100) according to claim 1, wherein the applicators include a temperature sensor (101), a pH sensor (102), a dissolved oxygen sensor (103), an acid applicator (114) a base applicator (115) and an air- ventilator (105).

8. A cell culture apparatus (100) according to claim 1 further comprising a stirrer (117) mounted on the covering means (120) and extended via an opening at the centre of the covering means (120) into the culture tank (110) for continuously stirring and mixing the cell culture in the culture tank (110).

9. A cell culture apparatus (100) according to claim 8 further comprising a motor (104) for rotating the stirrer (117).

10. A cell culture apparatus (100) according to claim 1 further comprising a supporting means (124) onto which the culture tank (110) is fitted.

11. A cell culture apparatus (100) according to claim 1 further comprising a viewing window (125) on the culture tank (110) allowing a user to observe the conditions of the cell cultures in the culture tank (110).

12. A cell culture apparatus (100) according to claim 1 is integrated with a computer program for monitoring and controlling the conditions in the culture tank (110).

13. A cell culture apparatus (100) according to claim 12, wherein the conditions in the culture tank (110) are controlled via a control panel (200).