CN115975794A - A device for automatic continuous cycle cell culture and treatment and its operation method - Google Patents

Abstract

The invention relates to the field of biological automation, in particular to a device for automatic continuous multi-cycle cell culture, cell induction, competent cell preparation and cell transformation and an operation method thereof. The device comprises the following modules: the cell culture module is used for culturing cells to a set cell concentration or culture time value; a cell induction module for inducing a cell to express a protein or molecule; a cell washing and concentrating module for preparing competent cells; the cell transformation module is used for efficiently transforming the exogenous substances into competent cells; a cleaning module for cleaning the containers of the modules; and the control module is used for sending instructions input by a user in advance to each module and each pump. The device has the advantages that the modules are connected in series to form a closed loop, the operation pollution risk is low, the micro-volume cell culture, the on-line cell concentration measurement and the continuous multi-round cell electroporation transformation can be realized, the operation is simple, the cost is low, and the device is suitable for the automatic continuous circulating culture and transformation of different types of cells.

Description

A device for automatic continuous cycle cell culture and treatment and its operation method

technical field

The present invention relates to the field of biological automation, in particular to a device for automatic continuous cycle cell culture and treatment and its operation method, more specifically to a device for automatic continuous multiple rounds of cell culture, cell induction, competent cell Devices for preparation, cell transformation and cell recovery and methods for their operation.

Background technique

An important step in genetic engineering technology is to introduce the target gene into the recipient cells. The conventional method is to manually inoculate the cells into a test tube or a shaker flask containing a medium in an ultra-clean table, and transfer them to a shaker. To a specific concentration, prepare the competent state. Competent preparation uses corresponding buffer or ultrapure water to wash and concentrate cells according to different preparation methods. This process requires repeated transfers between centrifuges and ultra-clean benches to centrifuge and resuspend cells, and then mix the target gene with Competent cells are mixed, transferred to a water bath for heat shock or transferred to an electroporation cup and placed in an electroporator for electric shock, and the treated cells are added to the medium for recovery. The revived cells are spread on solid plates or undergo the next gene transfer process. For example, multiplex automated genome engineering (MAGE) requires a dozen to dozens of consecutive cycles of gene introduction, which is time-consuming and labor-intensive for manual operation.

The Church research group has developed a device for introducing multiple nucleic acid sequences into cells, and all the steps of cell culture, competent preparation and electroporation transformation are completed in a custom-made electroporation cup (MULTIPLEX AUTOMATED GENOME ENGINEERING, Patent No. : WO2008052101A2). Although the device is simple, the completion of all operations in one unit increases the risk of bacterial contamination, and the accumulation of impurities also increases the difficulty of multiple rounds of transformation. However, because Cisco Ruipter has developed an automated multi-module cell editing instrument to automate genome editing in living cells, the device implements steps such as cell culture, competent preparation, and transformation in different modules. Transfer between modules (including automated cell processing methods, modules, instruments and systems for flow-through electroporation equipment, patent number: CN111386334 A). The device has the problems of complex system, large device volume, and increased contamination risk during sample transfer.

Electroporation transformation is currently the most efficient and most commonly used transformation method. Competent cells need to be prepared before electroporation transformation. Traditional electroporation competent cells are prepared by centrifuging cells in a high-speed centrifuge, resuspending cells in ultrapure water, and repeating many times. Washing and concentrating cells, the centrifuge method is difficult to realize automatic control, the present invention uses the membrane filtration method to realize cell washing and concentration, the membrane filtration method controls the liquid input and output cell suspension respectively by controlling different syringe pumps, and the liquid enters and exits through the filter membrane, reducing Cell loss, repeated several times to achieve automatic cell competent preparation. Conventional electroporation transformation is completed in the electroporation cup, which is a "static" transformation method, usually a one-time process, and it is difficult to achieve continuous multiple rounds of transformation. The present invention designs and processes microfluidic cell transformation chips. The voltage is applied during the process of exogenous substance mixture flowing through the chip channel to realize cell transformation, and the chip channel used can carry out multiple rounds of transformation continuously after automatic cleaning.

Contents of the invention

In view of this, the present invention provides a device for automatic continuous cycle cell culture and treatment and an operation method thereof. The device divides the steps of cell culture, cell induction, competent cell preparation and cell electroporation transformation into independent modules. The modules are connected in series to form a closed structure. The automatic operation of the modules is realized through the control program, which is especially suitable for different types of cells. Automated continuous cycle culture and transformation. The risk of contamination during the operation of the device is small, micro-cell culture can be realized, changes in cell concentration can be measured online in real time, and multiple rounds of continuous cell electroporation can be performed. The device is easy to operate and low in cost.

In order to achieve the above-mentioned purpose of the invention, the present invention provides a device for automatic continuous cycle cell culture and treatment and its operation method. Time data; cell induction module, used to induce cells to express certain proteins or molecules; cell washing and concentration module, used to prepare competent cells; cell transformation module, used to efficiently transform foreign substances into competent cells; cleaning module, connected to the cell culture module, cell induction module, cell washing and concentration module, and cell transformation module through valves, pumps, and pump tubes, respectively, for cleaning the cell culture module, cell induction module, cell washing and concentration module, and cell Part of the container of the transformation module; the control module is connected with the cell culture module, cell induction module, cell washing and concentration module, cell transformation module, cleaning module and pump respectively, and is used to send user pre-input instructions to each module and each pump , used to control the automatic operation of the whole device.

The cell culture module, the cell induction module, the cell washing and concentration module and the cell conversion module are sequentially connected in series through valves, pumps and pump tubes to form a closed loop.

The cell culture module includes at least one cell culture bottle for the first round of inoculating fresh cell liquid or receiving cell liquid from the cell transformation module.

In some specific embodiments of the present invention, the cell culture flask can be made of transparent glass or transparent plastic.

In some specific embodiments of the present invention, the cell culture flask may be circular, square or polygonal.

The cell culture module also includes a circuit board for receiving instructions sent by the control module, monitoring cell culture bottle data, and sending instructions to the control module.

The cell culture module also includes a stirring device for rotating the cell liquid in the cell culture bottle.

In some specific embodiments of the present invention, the stirring device includes a magnetic rotor, a heat dissipation fan, and a magnet attached to the blade of the heat dissipation fan, and the rotation of the magnet drives the rotation of the magnetic rotor in the bottle, thereby causing the cell fluid to stir.

The cell culture module also includes a temperature control device for controlling the temperature of the cell liquid in the cell culture flask.

In some specific embodiments of the present invention, the temperature control device includes a sleeve wrapping the cell culture flask, a temperature sensor and a heating resistor.

Preferably, the sleeve is made of a heat-conducting metal material, and the inner wall of the sleeve is matte-treated.

The cell culture module also includes an optical device for real-time measurement of cell concentration changes in the cell culture flask.

In some embodiments of the present invention, the optical device includes LEDs and photodiodes.

Preferably, the wavelength of the LED is 600-950nm.

The cell culture module also includes at least one culture medium bottle containing culture medium, a joint, a pump, a valve and a pump tube connecting the culture medium bottle to the cell culture bottle.

The cell culture module also includes a connector, a pump, a valve and a pump tube connecting the cell culture flask to the cell induction module.

The cell culture module also includes a connector, a pump, a valve and a pump tube connecting the cell culture flask to the cell transformation module.

The cell culture module also includes a connector, a pump, a valve and a pump tube connecting the cell culture bottle to the cleaning module.

The cell culture module also includes a joint, a pump, a valve and a pump tube for discharging the liquid in the cell culture bottle.

The culture mode of the cell culture module includes time mode and absorbance mode.

The time mode means that the system will automatically start the next step after running to the set time value.

In the absorbance mode, the next step of the program is automatically started when the cell growth reaches the set absorbance value.

The cell induction module comprises at least one culture bottle for receiving cell liquid from the cell culture bottle of the cell culture module.

In some specific embodiments of the present invention, the culture bottle can be made of transparent glass or transparent plastic.

Preferably, the culture bottle is made of glass material which is easier to conduct heat.

In some specific embodiments of the present invention, the culture flask may be round, square or polygonal.

The cell induction module also includes a circuit board for receiving instructions sent by the control module, monitoring culture bottle data, and sending instructions to the control module.

In some specific embodiments of the present invention, the cell induction module further includes a stirring device for rotating the cell liquid in the cell culture bottle.

In some specific embodiments of the present invention, the stirring device of the cell induction module includes a magnetic rotor, a heat dissipation fan, and a magnet attached to the blade of the heat dissipation fan, and the rotation of the magnet drives the rotation of the magnetic rotor in the bottle, thereby causing the cell fluid to rotate. Stir.

The cell induction module also includes a temperature control device for controlling the temperature of the cell liquid in the culture flask.

In some specific embodiments of the present invention, the temperature control device of the cell induction module includes a sleeve wrapping the culture bottle, a temperature sensor and a heating resistor.

Preferably, the sleeve is made of metal material that is easy to conduct heat.

In some specific embodiments of the present invention, the cell induction module further includes an optical device for real-time measurement of changes in cell concentration in the cell culture flask.

In some specific embodiments of the present invention, the optical device of the cell induction module includes LEDs and photodiodes.

The cell induction module also includes a connector, a pump, a valve and a pump tube connecting the culture flask to the cell washing and concentrating module.

The cell induction module also includes a connector, a pump, a valve and a pump tube connecting the culture flask to the cleaning module.

The cell induction module also includes a joint, a pump, a valve and a pump tube for discharging the liquid in the culture bottle.

The induction modes of the cell induction module include time mode and absorbance mode.

The cell washing and concentrating module includes at least one Erlenmeyer flask for receiving the cell solution from the culture bottle of the cell induction module, four syringe pumps (the first syringe pump, the second syringe pump, the third syringe pump, the Four syringe pumps), quick connectors, filters with membranes, and four-way joints connected to the first, second, and third syringe pumps and filters with membranes respectively.

The first syringe pump pumps 4°C sterile ultrapure water through the filter into the cell suspension in the Erlenmeyer flask, the second syringe pump pumps the suspension out of the Erlenmeyer flask through the filter, and the cells are trapped in the filter. On the membrane, the third syringe pump pumps sterile air into the Erlenmeyer flask through the filter, so that the cells trapped on the filter membrane enter the cell suspension, and the first syringe pump and the second syringe pump operate sequentially in a cycle. Wash and concentrate the cell solution in the shaped bottle.

The fourth syringe pump pumps the exogenous substance to be transformed into the washed and concentrated cell fluid.

In some specific embodiments of the present invention, the cell washing and concentrating module further includes a cooling chip for controlling the temperature of the Erlenmeyer flask.

In some specific embodiments of the present invention, the cell washing and concentrating module further includes a stirring device for rotating the cell liquid in the Erlenmeyer flask.

In some specific embodiments of the present invention, the stirring device of the cell washing and concentrating module includes a magnetic rotor, a heat dissipation fan, and a magnet attached to the blades of the heat dissipation fan.

In some specific embodiments of the present invention, the cell washing and concentrating module further includes a membrane washing step. The membrane washing step is to run the first syringe pump, the second syringe pump and the third syringe pump in sequence to wash the filter membrane on the filter.

The cell washing and concentrating module also includes joints, pumps, valves and pump tubes connected to the cell transformation module.

The cell washing and concentrating module also includes joints, pumps, valves and pump tubes connected to the cleaning module.

In some specific embodiments of the present invention, the cell washing and concentrating module further includes a joint, a pump, a valve and a pump tube for discharging the liquid in the Erlenmeyer flask.

The cell culture module, cell induction module, cell washing and concentration module all have waste liquid discharge process by default, that is, all the remaining cell liquid or washing liquid in the bottle will be discharged after each step of operation.

The cell transformation module includes a microfluidic cell transformation chip, a high-voltage amplifier, metal tubes, and plastic tubes.

The fabrication material of the microfluidic cell transformation chip includes polymethylmethacrylate, polydimethylsiloxane, ceramics, glass or other non-conductive materials.

The microfluidic cell transformation chip can be subjected to one or more sterilization methods, including high temperature sterilization, ultraviolet sterilization, and ethanol sterilization.

The microfluidic cell transformation chip includes at least one fluid channel, a channel inlet and a channel outlet.

The width, length and depth of the fluid channel of the microfluidic cell transformation chip can be adjusted according to different cell types.

In some specific embodiments of the present invention, the width of the fluid channel of the microfluidic cell transformation chip is 50-500 microns.

In some specific embodiments of the present invention, the fluid channel of the microfluidic cell transformation chip has a depth of 50-200 microns.

The metal tubes are respectively inserted into the fluid channel inlet and the channel outlet of the microfluidic cell transformation chip. The metal tubes are respectively connected to the high-voltage output terminal and the ground terminal of the high-voltage amplifier.

The plastic tubes are respectively connected to the metal tubes, and the mixture containing cells and foreign substances from the Erlenmeyer flask of the cell washing and concentration module is pumped into the fluid channel of the microfluidic cell transformation chip through a peristaltic pump.

Preferably, the high-voltage amplifier is started before the liquid or cells enter the fluid channel of the microfluidic cell transformation chip, and the high-voltage amplifier is turned off after all the cells flowing out of the fluid channel enter the cell culture flask of the cell culture module.

The cells flowing out of the microfluidic cell transformation chip of the cell transformation module flow into the cell culture bottle of the cell culture module through the pump tube for the next round of cell culture and treatment.

The cleaning module includes at least one cleaning bottle, a connector for discharging liquid in the bottle, a pump, a valve and a pump tube.

In some specific embodiments of the present invention, the cleaning module includes a cleaning bottle filled with sterilized ultrapure water, a cleaning bottle filled with 75% ethanol, and the cell culture bottle of the cell culture module, the culture bottle of the cell induction module, The joints, pumps, valves and pump tubes that are connected to the Erlenmeyer flask of the concentration module and the fluid channel of the microfluidic cell transformation chip of the cell transformation module are washed by the cells.

In some specific embodiments of the present invention, the liquid in the cleaning bottle of the cleaning module is sterilized ultrapure water.

In some specific embodiments of the present invention, the liquid in the cleaning bottle of the cleaning module is 75% ethanol.

The control module includes a controller and a control circuit.

The controller includes at least an automatic mode program, a manual mode program, and an absorbance (OD600) calibration program.

An operation method flow chart of a device for automatic continuous cycle cell culture and treatment is as follows:

(1) As a preference, at first the absorbance curve of the instrument is calibrated. The calibration process is to use a commercially available spectrophotometer to measure the absorbance (OD600) value of the freshly cultured cell fluid, and dilute the OD600 value of the cell fluid to 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, the volume of each dilution should not be less than 3mL. Turn on the absorbance (OD600) calibration program, transfer the dilutions to the cell culture flasks in sequence, and record the light intensity values read by the instrument in the program. After all readings, the instrument automatically draws a standard curve. During the cell growth process, the instrument automatically updates the OD600 value of cell growth every 10 seconds according to the drawn curve and the light intensity value read by the instrument.

(2) The bottles and pump tubes of the cell culture module, cell induction module and cleaning module of the instrument device are sterilized at high temperature, and the cell washing and concentration module and cell transformation module are sterilized with 75% ethanol.

(3) After sterilization, the device is assembled in the ultra-clean bench, and a certain volume of fresh cell culture solution is inserted into the culture bottle of the cell culture module.

(4) Turn on the automatic program of the controller, and set the culture mode, culture temperature, stirring speed, culture medium volume, transfer volume (that is, the volume of cell liquid transferred to the next unit) and washing liquid volume of the cell culture module.

(5) Set the induction mode, induction temperature, stirring speed, transfer volume (that is, the volume of the cell fluid transferred to the next unit) and the volume of washing solution of the cell induction module.

(6) Set the operating temperature, stirring speed, pumping volume, pumping speed, discharge speed, and washing liquid volume of each pump for the cell washing and concentration module.

(7) Set the high pressure cycle and high pressure duty cycle of the cell transformation module, and the operating speed of the pump.

(8) Set the number of cycles and issue an instruction to start the device.

Due to the adoption of the above technical scheme, the present invention has the following advantages: the device fully automates the entire process of cell culture, cell induction, competent cell preparation, cell transformation and cell recovery, and the entire process can run continuously for multiple cycles; Participating in large-scale devices, the inventive device is more miniaturized and easier to operate; compared with the traditional electro-cup transformation method, the microfluidic chip cell flow electrotransformation of the inventive device can be realized continuously for multiple rounds and the transformation efficiency is higher.

Description of drawings

Fig. 1 is a schematic diagram of the overall external structure design of the automation system of the present invention.

Fig. 2a is a schematic design diagram of the cell culture module 1 provided by the embodiment of the present invention.

Fig. 2b is a schematic diagram of the positions of the optical elements for measuring cell growth in the cell culture module 1 provided by the embodiment of the present invention.

Fig. 2c is a curve diagram of calibration of the light intensity measured by the cell culture module 1 to the OD600 measured by the spectrophotometer provided in the embodiment of the present invention.

Fig. 2d is the growth curve of Escherichia coli cells measured in the customized cell culture flask by the cell culture module 1 provided in the embodiment of the present invention, and compared with the growth of the 100mL shake flask.

Figure 2e is the growth curve of Escherichia coli within 0-3 hours before Figure 2d.

Fig. 3a is a schematic diagram of the design of the cell induction module 2 provided by the embodiment of the present invention.

Figure 3b shows the growth of Escherichia coli cells grown to OD600=0.4 in the cell culture module 1 and continued to induce culture at 42°C in the cell induction module 2 for different time periods provided by the embodiment of the present invention.

Fig. 3c shows the homologous recombination efficiency of E. coli cells induced and cultured at 42°C for different time periods in the cell induction module 2 of Example 3 of the present invention.

Fig. 4a is a schematic diagram of the structural design of the cell washing and concentrating module 3 of the embodiment of the present invention.

Fig. 4b shows the volume of competent cells and the amount of remaining cells prepared by continuous treatment of 8 cycles of the single filter of the cell washing and concentration module 3 provided in Example 4 of the present invention.

Fig. 5a is a schematic diagram of the microfluidic cell transformation chip cell flow electroporation transformation provided in Example 5 of the present invention.

Fig. 5b is a schematic diagram of the channel structure of the microfluidic cell transformation chip provided in Example 5 of the present invention.

Fig. 5c shows the effect of the channel width of the microfluidic cell transformation chip provided in Example 5 of the present invention on the cell transformation efficiency.

Fig. 5d is the effect of the channel depth of the microfluidic cell transformation chip provided in Example 5 of the present invention on the cell transformation efficiency.

Fig. 6 is a schematic structural view of the cleaning module 5 according to the embodiment of the present invention.

Fig. 7 is a schematic structural diagram of the control module 6 according to the embodiment of the present invention.

In the figure: 1. Cell culture module; 2. Cell induction module; 3. Competent preparation module; 4. Cell transformation module; 5. Cleaning module; 6. Control module; 10. Culture medium bottle; 101. Culture medium; 11 , culture bottle; 121, casing; 122, circuit board; 131, temperature sensor; 132, heating resistor; 141, LED; 142, photodiode; 151, magnetic rotor; 152, cooling fan; 153, magnet; 21, cultivation bottle; 221, casing; 222, circuit board; 231, temperature sensor; 232, heating resistor; 241, LED; 242, photodiode; 251, magnetic rotor; 252, cooling fan; 253, magnet; 31, Erlenmeyer flask ; 321, the first injection pump; 322, the second injection pump; 323, the third injection pump; 324, the fourth injection pump; 33, the filter; 34, the cooling plate; 351, the magnet; 352, the magnetic rotor; 353, the cooling Fan; 40. Peristaltic pump; 41. Microfluidic electroporation conversion chip; 42. High-voltage amplifier; 43. Stainless steel tube; 44. Silicone tube; 45. Cell and nucleic acid mixture; 51. Ultrapure water; 52. 75% ethanol ; 53, pump and pump tube; 61, controller; 62, control circuit board.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1

With reference to FIG. 1 , a device for automated continuous cycle cell culture and treatment provided in this embodiment includes: a cell culture module 1 for culturing cells to grow to a user-set cell concentration or culture time data; a cell induction module 2, used to induce cells to express certain proteins or molecules; cell washing and concentration module 3, used to prepare electroporation transformation competent cells; cell transformation module 4, used for electroporation transformation competent cells; cleaning module 5, used for Part of the containers of the cell culture module, cell induction module, cell washing and concentration module and cell transformation module are cleaned; the control module 6 is used to control the operation of the entire program.

The cell culture module 1, the cell induction module 2, the cell washing and concentration module 3 and the cell transformation module 4 are sequentially connected in series through a one-way valve, a peristaltic pump and a pump tube to form a closed loop.

The cell culture module 1 includes at least one cell culture bottle 11 for inoculating fresh cell liquid in the first round or receiving cell liquid from the cell transformation module. The cell culture bottle 11 is made of transparent glass or transparent plastic, and its shape can be Circular, square or polygonal.

The cell culture module 1 also includes a temperature control device for controlling the temperature of the cell liquid in the cell culture flask. The temperature control device includes a sleeve 121 wrapping the cell culture bottle 11 , a temperature sensor 131 and a heating resistor 132 . Preferably, the sleeve 121 is made of metal material that is easy to conduct heat, and the inner wall of the sleeve is matte treated.

The cell culture module 1 also includes a circuit board 122 for receiving instructions sent by the control module 6 , monitoring data of the cell culture bottle 11 and sending instructions to the control module 6 .

The cell culture module 1 also includes a stirring device for rotating the cell liquid in the cell culture bottle. The stirring device includes a magnetic rotor 151 , a heat dissipation fan 152 and a magnet 153 adhered to the blade of the heat dissipation fan 153 . .

The cell culture module 1 also includes an optical device for real-time measurement of changes in cell concentration in the cell culture flask. Specifically, the optical device includes an LED 141 and a photodiode 142. The wavelength of the LED141 is 600-950nm.

The cell culture module 1 also includes at least one medium bottle 10 containing a medium 101 , a joint connecting the medium bottle to the cell culture bottle 11 , a peristaltic pump, a one-way valve and a pump tube.

The cell culture module 1 also includes a joint, a peristaltic pump, a one-way valve and a pump tube connecting the cell culture bottle 11 to the cell induction module 2; A joint, a peristaltic pump, a one-way valve and a pump tube connected to the module 4; a joint, a peristaltic pump, a one-way valve and a pump tube connecting the cell culture bottle 11 to the cleaning module 5 are also included; The connector, peristaltic pump, one-way valve and pump tube for liquid discharge in the cell culture bottle 11.

The culture mode of the cell culture module 1 includes time mode and absorbance mode.

The time mode means that the system will automatically start the next step after running to the set time value.

In the absorbance mode, the next step of the program is automatically started when the cell growth reaches the set absorbance value.

The cell induction module 2 includes at least one culture bottle 21 for receiving cell liquid from the cell culture bottle 11 . The culture bottle 21 can be made of transparent glass or transparent plastic. Preferably, the culture bottle 21 is made of glass material that is more likely to conduct heat, and its shape can be round, square or polygonal.

The cell induction module 2 also includes a circuit board 222 for receiving instructions sent by the control module 6, monitoring the data of the culture bottle 21 and sending instructions to the control module.

The cell induction module 2 also includes a stirring device for rotating the cell liquid in the culture bottle 21 . Specifically, the stirring device includes a magnetic rotor 252 , a heat dissipation fan 253 and a magnet 251 adhered to the blades of the heat dissipation fan 253 .

The cell induction module 2 also includes a temperature control device for controlling the temperature of the cell liquid in the culture bottle 21 . Specifically, the temperature control device includes a sleeve 221 wrapping the culture bottle 21 , a temperature sensor 231 and a heating resistor 232 . Preferably, the sleeve 221 is made of metal material that is easy to conduct heat.

The cell induction module 2 also includes an optical device for measuring the change of the cell concentration in the culture flask 21 in real time. Specifically, the optical device includes an LED 241 and a photodiode 242 .

The cell induction module 2 also includes a joint, a peristaltic pump, a one-way valve and a pump tube connecting the culture bottle 21 to the cell washing and concentration module 3; it also includes connecting the culture bottle 21 to the cleaning module 3 connected connectors, peristaltic pumps, one-way valves and pump tubes; also include connectors, peristaltic pumps, one-way valves and pump tubes for discharging the liquid in the culture bottle 21.

The induction modes of the cell induction module 2 include time mode and absorbance mode.

The cell washing and concentrating module 3 includes an Erlenmeyer flask 31 for receiving the cell solution from the culture bottle 21, four syringe pumps (i.e. the first syringe pump 321, the second syringe pump 322, the third syringe pump 323, the fourth syringe pump Pump 324), four-way joint 325, filter 33 with filter membrane.

The four-way connector 325 is respectively connected with the first syringe pump 321 , the second syringe pump 322 , the third syringe pump 323 and the filter 33 .

The first syringe pump 321 pumps 4°C sterile ultrapure water into the cell suspension in the Erlenmeyer flask 31 through the filter 33, and the second syringe pump 322 pumps the suspension out of the Erlenmeyer flask 31 through the filter 33, The cells are trapped on the filter membrane of the filter 33, and the third syringe pump 323 pumps sterile air into the Erlenmeyer flask 31 through the filter 33, so that the cells trapped on the filter membrane enter the cell suspension, and the first syringe pump 321 , the second syringe pump 322 runs sequentially in a cycle to wash and concentrate the cell solution in the Erlenmeyer flask 31 .

The fourth syringe pump 324 pumps the exogenous substance to be converted into the washed and concentrated cell fluid.

The cell washing and concentrating module 3 also includes a cooling chip 34 for controlling the temperature of the Erlenmeyer flask 31 .

The cell washing and concentrating module 3 also includes a stirring device for rotating the cell liquid in the Erlenmeyer flask 31 . Specifically, the stirring device includes a magnetic rotor 352, a heat dissipation fan 353, and a magnet 351 adhered to the blade of the heat dissipation fan 353.

The cell washing and concentrating module 3 also includes a membrane washing step. In the membrane washing step, the first syringe pump 321 , the second syringe pump 322 and the third syringe pump 323 are used to cycle in sequence to wash the filter membrane on the filter 33 .

The cell washing and concentrating module 3 also includes a joint for discharging the liquid in the Erlenmeyer flask 31, a peristaltic pump, a one-way valve and a pump tube.

The cell culture module 1, the cell induction module 2, and the cell washing and concentration module 3 all have a waste liquid discharge process by default, that is, the cell culture bottle 11 of the cell culture module 1 and the cell induction module 2 are cultured after the operation of the modules is completed. The remaining cell liquid or washing liquid in the flask 21 and the Erlenmeyer flask 31 of the cell washing and concentrating module 3 are all discharged.

The cell transformation module 4 includes a microfluidic cell transformation chip 41 , a high-voltage amplifier 42 , a metal tube 43 and a plastic tube 44 .

The fabrication material of the microfluidic cell transformation chip 41 includes polymethylmethacrylate, polydimethylsiloxane, ceramics, glass or other non-conductive materials. The microfluidic cell transformation chip 41 can be subjected to one or more sterilization methods, including high temperature sterilization, ultraviolet sterilization, and ethanol sterilization. The microfluidic cell transformation chip 41 includes at least one fluid channel 411 , a channel inlet 412 and a channel outlet 413 . The width, length and depth of the fluid channel 411 of the microfluidic cell transformation chip 41 can be adjusted according to different cell types.

The metal tubes 43 are respectively inserted into the fluid channel inlet 412 and the channel outlet 413 . The metal tubes 43 are respectively connected to the high voltage output end and the ground end of the high voltage amplifier 42 .

The plastic tubes 44 are respectively connected to the metal tubes 43, and the mixture containing cells and foreign substances from the Erlenmeyer flask 31 of the cell washing and concentration module 3 is pumped into the fluid channel 411 of the microfluidic cell transformation chip 41 through the peristaltic pump 40 .

The opening of the high-voltage amplifier 42 is before the operation of the peristaltic pump that controls the mixture of cells and foreign substances entering the chip channel 411, and the closing of the high-voltage amplifier 42 means that all the cell fluid flowing out of the fluid channel 411 enters the cell culture module 1. After bottle 11.

The cells flowing out of the microfluidic cell transformation chip 41 of the cell transformation module 4 flow into the cell culture bottle 11 of the cell culture module 1 through the pump tube for the next round of cell culture and treatment.

The cleaning module 5 includes a cleaning bottle 51 containing 75% ethanol, a cleaning bottle 52 containing sterilized ultrapure water, and connecting the cleaning bottles 51, 52 with the cell culture bottle 11 of the cell culture module 1 and the cell induction module 2. Culture flask 21 , Erlenmeyer flask 31 of cell washing and concentrating module 3 , joint connected to fluid channel 411 of microfluidic cell transformation chip 41 of cell transformation module 4 , peristaltic pump, one-way valve and pump tube.

The operation of the cleaning module 5 is to use the cleaning solution in the cleaning bottle 51 and the cleaning bottle 52 to wash each module container successively after the operation of each module connected to it is completed, that is, after the cell culture module 1 runs, use 75 of the cleaning bottle 51 to clean the container. % ethanol and ultrapure water in the cleaning bottle 52 to clean the cell culture bottle 11 of the cell culture module 1, the culture bottle 21 of the cell induction module 2, the Erlenmeyer flask 31 of the cell washing and concentration module 3, and the microfluidics of the cell transformation module 4 The cleaning of the control cell transformation chip 41 is also the same.

The control module 6 includes a controller 61 and a control circuit 62 . The controller 61 includes at least an automatic mode program, a manual mode program, and an absorbance (OD600) calibration program. The control module 6 is respectively connected with the cell culture module 1, the cell induction module 2, the cell washing and concentration module 3, the cell transformation module 4, the cleaning module 5, and the peristaltic pump, and is used to send user pre-input instructions to each module and peristaltic pump.

Example 2

The automatic cultivation of Escherichia coli is realized by using the cell culture module 1 of the automatic continuous cycle cell culture and treatment device provided by the present invention. FIG. 2 a is a schematic diagram of the design of the cell culture module 1 . The cell culture module 1 of this embodiment uses a custom-made square bottle as the culture bottle 11. The inner side length of the square bottle is 19 mm, the inner width is 12 mm, and the volume is 10 mL. Figure 2b shows the position of the optical elements when culturing cells in square culture flasks.

Use a commercially available spectrophotometer to measure the absorbance (OD600) value of the freshly cultivated Escherichia coli cell liquid, and dilute the OD600 value of the cell liquid to 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, the volume of each dilution should not be less than 3mL. Turn on the instrument absorbance (OD600) calibration program, transfer the dilutions to the cell culture bottle 11 in sequence, and record the light intensity value read by the instrument in the program. After all readings, the instrument automatically draws a standard curve. Figure 2c is the calibration of the light intensity of different concentrations of Escherichia coli measured by the cell culture module 1 to the OD600 value measured by the spectrophotometer. During the culture of E. coli cells, the instrument automatically updates the OD600 value of E. coli cell growth every 10 seconds according to the drawn curve and the light intensity value read by the instrument. 100 microliters of fresh cell culture solution with OD600=3 was placed in the culture bottle 11 of the cell culture module 1, the volume of the culture medium 101 was 3 mL, and the culture temperature was 30°C. Figure 2d is a comparison of the growth of E. coli in custom cell culture flask 11 and 100mL shake flask. Figure 2e shows the cell growth situation specific to the first 0-3 hours in Figure 2d.

Example 3

Automatic cell induction is realized by using the cell induction module 2 of the automatic continuous cycle cell culture and treatment device provided by the present invention. Fig. 3a shows a schematic diagram of the design of the cell induction module 2 provided in this embodiment. The Escherichia coli ATCC 8739 (pORTMAGE) cells of Example 2 were cultivated to 600=0.4, and the automatic control syringe pump transferred 2mL of the cell solution to the culture bottle 21 of the cell induction module 2, and the culture bottle 21 of the present embodiment was a 20mL screw-top black cap bottle. The cell fluid was cultured at 42°C for different time to induce the expression of the recombinant enzyme. Wherein the plasmid pORTMAGE is derived from the reference Nyerges et al, Proc NatlAcad Sci USA.2016,113(9):2502-7.

Take 1mL of cell fluid and centrifuge and wash to prepare competent cells, electrotransform the artificially synthesized ssDNA containing the gene lacZ expression that terminates in advance into competent cells, and revive the cells coated with IPTG (isopropylthio-β-D-galactoside ) and X-gal (5-bromo-4-chloro-3-indole-β-D-galactoside) LB solid plate, wait for a single colony to grow, count the number of white colonies. Figure 3b shows the growth of cells cultured at 42°C for different time periods. Figure 3c shows the proportion of white colonies, that is, the efficiency of homologous recombination, for different times of induction culture at 42°C.

Example 4

The automatic competent cell preparation is realized by using the cell washing and concentration module 3 of the automatic continuous cycle cell culture and treatment device provided by the present invention. After incubating the cell solution of Example 3 at 42° C. for 15 minutes, transfer 1 mL of the cell solution to the Erlenmeyer flask 31 of the cell washing and concentration module 3 . Fig. 4a is a schematic structural diagram of the cell washing and concentrating module 3 according to the embodiment of the present invention.

The four syringe pumps are continuous syringe pumps with multi-channel valve heads. One valve head of the first syringe pump 321 is connected to ultrapure water, and the other valve head is connected to the four-way connector 325; one valve head of the second syringe pump 322 is connected to the four-way connector 325, and the other valve head is connected to the waste liquid container; the third valve head is connected to the four-way connector 325; One valve head of the syringe pump 323 is connected to the filter 33 with a 0.2 μm filter membrane, and the other valve head is connected to the four-way joint 325; the fourth syringe pump 324 has one valve head connected to the Erlenmeyer flask for storing ssDNA, and the other valve head is connected to the conical Culture bottle 31; set the first syringe pump 321 with a liquid inlet flow rate of 5 s/mL, a liquid discharge flow rate of 10 s/mL, a volume of ultrapure water of 1 mL, a pump 322 with a liquid inlet flow rate of 30 s/mL, a liquid discharge flow rate of 5 s/mL, and a liquid discharge volume of 1.15mL, pump 321 and pump 322 cycled 10 times, pump 323 inlet flow rate 5s/mL, exhaust flow rate 10s/mL, sterile air volume 2mL, pump 324 inlet flow rate 15s/mL, discharge flow rate 15s /mL, the volume of ssDNA is 0.05mL.

After each run, record the remaining cytosol volume and cell mass. In addition, there is a membrane washing step after each operation, that is, the first syringe pump 321 and the third syringe pump 323 cycle for 3 times, and then enter the next competent state preparation process.

Each filter was run 8 times. Figure 4b shows the preparation of the competent volume and the amount of remaining cells for 8 cycles of continuous processing of the single filter of the cell washing and concentration module 3.

Example 5

Cell transformation is achieved by using the cell transformation module 4 of the automatic continuous cycle cell culture and treatment device provided by the present invention. In the present invention, the cell flow electroporation transformation based on the microfluidic chip is used to replace the electroporation using the electroporation cup in the laboratory, and the working diagram of the microfluidic cell transformation chip 41 cell flow transformation is shown in Figure 5a. The microfluidic cell transformation chip 41 of this embodiment is formed by bonding polydimethylsiloxane and glass, the fluid channel 411 of the chip is a parallel channel, the channel length is 3000 microns, and the length from the inlet to the outlet of the chip is 5800 microns. The chip channel structure is shown in Figure 5b. The competent cells prepared in Example 4 were injected into microfluidic cell transformation chips with different depths and widths at a rate of 200 μL/min, and the electroporation method was used as a control. The results are shown in Figures 5c and 5d. When it is 100 microns, the conversion efficiency is the highest, and both are better than the control of the electro-cup method.

Example 6

The flow chart of the device operation method for automatic continuous cycle cell culture and treatment in the above embodiments of the present invention is as follows:

1) First, calibrate the absorbance curve of the instrument. The specific calibration process is to use a commercially available spectrophotometer to measure the absorbance (OD600) value of the freshly cultured cell fluid, and then dilute the OD600 value of the cell fluid to 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4 , 1.6, 1.8, 2, the volume of each dilution shall not be less than 3mL. Turn on the absorbance (OD600) calibration program, transfer the dilutions to the cell culture bottle 11 in sequence, and record the light intensity value read by the instrument in the program. After all readings, the instrument automatically draws a standard curve. During the cell growth process, the instrument automatically updates the OD600 value of cell growth every 10 seconds according to the drawn curve and the light intensity value read by the instrument.

2) Carry out high-temperature sterilization to the cell culture bottle 11 of the cell culture module 1 of the device, the culture bottle 21 of the cell induction module 2, the cleaning bottle 51 of the sterilized ultrapure water of the cleaning module 5, and the pump tube, and cell washing and The Erlenmeyer flask 31 of the concentration module 3, the microfluidic cell transformation chip 41 of the cell transformation module 4, and the connecting pump tube are sterilized with 75% ethanol.

3) After sterilization, the whole device is assembled in a sterile ultra-clean bench, and a certain volume of fresh cell culture solution is inserted into the cell culture bottle 11 of the cell culture module 1 .

4) Open the automatic mode program of the controller 61, set the culture mode of the cell culture module 1, the culture temperature, the volume of the culture medium, the transfer volume (the volume that transfers the cell solution to the next unit), and the washing solution of the washing bottle 51 to 75% The volume of each run of ethanol, the volume of each run of wash bottle 52 ultrapure water.

5) Set the induction mode of the cell induction module 2, the induction temperature, the transfer volume (i.e. the volume of the cell fluid transferred to the next unit), the volume of each operation of 75% ethanol in the cleaning bottle 51, and the ultrapure water in the cleaning bottle 52 each time. Operating volume.

6) Set the operating temperature of the cell washing and concentrating module 3 , the extraction volume, extraction speed, and discharge speed of the first syringe pump 321 , the second syringe pump 322 , the third syringe pump 323 , and the fourth syringe pump 324 .

7) Set the high pressure cycle and high pressure duty cycle of the cell transformation module 4, and the operating speed of the peristaltic pump 40.

8) Set the number of cycles and issue an instruction to start the device.

Claims (10)

1. An apparatus for automated continuous cycle cell culture and processing, comprising: it includes the following modules that are connected in series in proper order through valve, pump and pump line:

the cell culture module is used for culturing cells to a cell concentration or cell culture time value set by a user;

a cell induction module for inducing a cell to express a protein or molecule;

a cell washing and concentrating module for preparing competent cells;

the cell transformation module is used for efficiently transforming the exogenous substances into competent cells;

the cleaning module is respectively connected with the cell culture module, the cell induction module, the cell washing and concentrating module and the cell transformation module through a valve, a pump and a pump pipe and is used for cleaning containers of all the modules; the cleaning module comprises at least one cleaning bottle for containing cleaning liquid, a joint for discharging liquid in the bottle, a pump, a valve and a pump pipe;

and the control module is respectively connected with the cell culture module, the cell induction module, the cell washing and concentrating module, the cell transformation module, the cleaning module and the pump, and is used for sending a command which is input by a user in advance to each module and each pump and controlling the whole device to operate.

2. An apparatus for automated continuous cycle cell culture and processing according to claim 1, wherein: the cell culture module comprises at least one cell culture bottle (preferably, the cell culture bottle is made of transparent glass material and is circular, square or polygonal) for inoculating fresh cell liquid or receiving the cell liquid from the cell transformation module, the cell culture module further comprises a stirring device for rotating the cell liquid in the cell culture bottle, and/or a temperature control device for controlling the temperature of the cell liquid in the cell culture bottle, and/or an optical device for measuring the change of the cell concentration in the cell culture bottle in real time, the optical device preferably comprises an LED and a photodiode, and the wavelength of the LED is more preferably 600-950nm; the cell culture module, the cell induction module, the cell washing and concentration module and the cell transformation module are sequentially connected in series through a valve, a pump and a pump pipe to form a closed loop.

3. An apparatus for automated continuous cycle cell culture and processing according to claim 2, wherein: the cell culture module also comprises a circuit board for receiving the control module sending instruction, monitoring cell culture bottle data and sending the instruction to the control module.

4. An apparatus for automated continuous cycle cell culture and processing according to claim 3, wherein: the cell culture module also comprises at least one culture medium bottle for containing culture medium, and a joint, a pump, a valve and a pump pipe for connecting the culture medium bottle and the cell culture bottle; preferably, the connectors, pumps, valves and pump lines of the cell culture flask and the cell induction module, and/or the cell transformation module, and the cell culture module further comprises connectors, pumps, valves and pump lines for draining the liquid in the cell culture flask;

preferably, the culture mode of the cell culture module comprises a time mode and an absorbance mode; the time mode is that the system automatically starts the next procedure after running to the set time value; the absorbance pattern, i.e., the time when cell growth reaches a set absorbance value, automatically initiates the next procedure.

5. An apparatus for automated continuous cycle cell culture and processing according to claim 1, wherein: the cell induction module comprises at least one culture flask for receiving cell broth from the cell culture module; the circuit board is used for receiving the command sent by the control module, monitoring the data of the culture bottle and sending the command to the control module; preferably, the cell induction module further comprises a stirring device for rotating the cell fluid in the culture bottle, and/or further comprises a temperature control device for controlling the temperature of the cell fluid in the cell culture bottle, and/or further comprises an optical device for measuring the change of the cell concentration in the cell culture bottle in real time, and/or further comprises a connector, a pump, a valve and a pump pipe for connecting the culture bottle with the cell washing and concentrating module, and a connector, a pump, a valve and a pump pipe for discharging the liquid in the culture bottle;

optionally, the induction mode of the cell induction module comprises a temporal mode and an absorbance mode.

6. An apparatus for automated continuous cycle cell culture and processing according to claim 1, wherein: the cell washing and concentrating module comprises a conical flask for receiving cell liquid from the cell induction module, a first injection pump for controlling sterile ultrapure water, a second injection pump for controlling cell suspension, a third injection pump for controlling sterile air, a fourth injection pump for controlling a substance to be transferred into an exogenous substance, a filter with a filter membrane, a four-way joint respectively connected with the first injection pump, the second injection pump, the third injection pump and the filter with the filter membrane, and a quick-connection joint for connecting the filter and the four-way joint, wherein the first injection pump pumps the sterile ultrapure water into the cell suspension in the conical flask through the filter, the second injection pump pumps the cell suspension out of the conical flask through the filter, the third injection pump pumps the sterile air into the conical flask through the filter membrane, so that cells left on the filter membrane enter the cell suspension, and the fourth injection pump 3 pumps the exogenous substance to be converted into the washed and concentrated cell liquid. The cell washing and concentrating module further comprises a refrigerating sheet for controlling the temperature of the conical flask, and/or further comprises a stirring device for rotating cell liquid in the conical flask, preferably, the stirring device comprises a magnet, a magnetic rotor and a cooling fan; further preferably, the method also comprises a membrane washing step, wherein the membrane washing step is to wash the upper filter membrane of the filter by using a first injection pump, a second injection pump and a third injection pump to sequentially and circularly operate. Further comprises a connector, a pump, a valve and a pump pipe which are connected with the cell transformation module, and a connector, a pump, a valve and a pump pipe which are used for discharging the liquid in the conical flask.

7. An apparatus for automated continuous cycle cell culture and processing according to claim 1, wherein: the cell transformation module comprises a microfluidic cell transformation chip, a high-pressure amplifier, a metal tube and a plastic tube, wherein the plastic tube is respectively connected with the metal tube, a mixed fluid containing cells and exogenous substances from the cell washing and concentrating module is introduced into a fluid channel of the microfluidic cell transformation chip, the cells flowing through the chip channel flow into the cell culture module, the metal tube is respectively inserted into a fluid channel inlet and a channel outlet of the microfluidic cell transformation chip, and the metal tube is respectively connected with a high-pressure output end and a grounding end of the high-pressure amplifier; the micro-fluidic cell transformation chip also comprises a part for sterilizing the micro-fluidic cell transformation chip, wherein the sterilization is high-temperature sterilization, ultraviolet sterilization or ethanol sterilization;

preferably, the width, length and depth of the fluid channel of the microfluidic cell conversion chip can be adjusted, specifically, the width of the fluid channel of the microfluidic cell conversion chip is 50-500 micrometers, and the depth of the fluid channel of the microfluidic cell conversion chip is 50-200 micrometers;

preferably, the high-pressure amplifier is started before the liquid or the cells enter the fluid channel of the microfluidic cell conversion chip, and the high-pressure amplifier is closed after all the cells flowing out from the fluid channel enter the cell culture bottle of the cell culture module.

8. An apparatus for automated continuous cycle cell culture and processing according to claim 1, wherein: the cleaning module operates to use the cleaning liquid in the cleaning bottle to clean each module container after the operation of each module connected with the cleaning module is finished, namely the cell culture module, the culture bottle of the cell induction module, the conical flask of the cell cleaning and concentration module and the fluid channel of the microfluidic cell conversion chip of the cell conversion module;

the cell culture module, the cell induction module and the cell washing and concentrating module are all provided with programs for discharging residual cell sap or washing liquid in a cell culture bottle of the cell culture module, a culture bottle of the cell induction module and a conical flask of the cell washing and concentrating module after the operation is finished;

preferably, the cell sap flowing out of the fluid channel of the microfluidic cell conversion chip of the cell conversion module flows into the cell culture bottle of the cell culture module through the pump tube to perform the next round of cell culture and treatment.

9. An apparatus for automated continuous cycle cell culture and processing according to claim 1, wherein: the control module controller and the control circuit, the controller includes at least an automatic mode program, a manual mode program, and an absorbance calibration program.

10. A method of operating an apparatus for automated continuous cycle cell culture and processing according to any one of claims 1 to 9, wherein: the method comprises the following steps:

(1) Firstly, calibrating an absorbance curve of an instrument;

(2) Sterilizing a cell culture bottle of a cell culture module, a culture bottle of a cell induction module, a cleaning bottle of a cleaning module and a pump pipe of the instrument device at high temperature, and sterilizing a conical bottle of a cell washing and concentrating module and a fluid channel of a microfluidic cell conversion chip of a cell conversion module by using 75% ethanol for example;

(3) After sterilization, the device is assembled in a sterile super clean bench, and a certain volume of fresh cell culture solution is added into a culture bottle of the cell culture module;

(4) Opening an automatic mode program of the controller, and setting a culture mode, a culture temperature and a culture medium volume of the cell culture module, a transfer volume for transferring cell sap to the next module and a washing liquid volume;

(5) Setting an induction mode and an induction temperature of the cell induction module, a transfer volume for transferring the cell sap to the next module, and a washing liquid volume;

(6) Setting the operation temperature of the cell washing and concentrating module, the pumping volume, the pumping speed, the discharging speed and the washing liquid volume of each pump;

optionally, further comprising (7) setting the high-pressure period and the high-pressure duty ratio of the cell transformation module and the running speed of the pump;

(8) Setting cycle times and sending an instruction to start the device;

preferably, the absorbance curve calibration process is to measure the absorbance (OD 600) value of freshly cultured cell sap by using a commercial spectrophotometer, and sequentially dilute the OD600 value of the cell sap to 0,0.1,0.2,0.4,0.6,0.8,1,1.2,1.4,1.6,1.8,2, wherein each diluent has a volume of not less than 3mL; the absorbance (OD 600) calibration procedure was opened and the dilutions were sequentially transferred to cell culture flasks and the light intensity values read by the instrument were recorded in the procedure. After all the data are read, the instrument automatically draws a standard curve; during the cell growth process, the OD600 value of the cell growth is automatically updated by the instrument every 10 seconds according to the drawn curve and the light intensity value read by the instrument.

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