CN115975794A - Device for automatic continuous circulating cell culture and treatment and operation method thereof - 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

Device for automatic continuous circulating cell culture and treatment and operation method thereof

Technical Field

The invention relates to the field of biological automation, in particular to a device for automatic continuous circulating cell culture and treatment and an operation method thereof, and more particularly relates to a device for automatic continuous multi-round cell culture, cell induction, competent cell preparation, cell transformation and cell resuscitation and an operation method thereof.

Background

An important step of genetic engineering technology is to introduce the target gene into the receptor cell, and the conventional method is to manually inoculate the cell in a superclean bench into a test tube containing a culture medium or a shake flask, transfer the cell to a shaking table, during which a sample is taken to determine the change of the cell concentration, and culture the cell to a certain specific concentration to prepare competence. The competent preparation uses corresponding buffer solution or ultrapure water to wash and concentrate cells according to different preparation methods, the process needs to be repeatedly transferred between a centrifugal machine and a super clean bench for centrifugation and cell resuspension for many times, then the target gene and the competent cells are mixed, transferred to a water bath pot for heat shock or transferred to an electric rotating cup and placed in an electric rotating instrument for electric shock, and the treated cells are added into a culture medium for resuscitation. The recovered cells were coated on a solid plate or subjected to the next gene transfer process. For example, multiplex Automated Genome Engineering (MAGE) requires a gene transfer process of ten or more continuous cycles to several tens of cycles, and requires time and labor for manual operation.

The Church research group developed a device for introducing multiple nucleic acid sequences into cells that focused all steps of cell culture, competent preparation, and electroporation transformation into cells in a custom-made electroporation cuvette (WO 2008052101A 2). Although simple, all operations are carried out in one unit, which increases the risk of contamination, and in addition the accumulation of impurities also increases the difficulty for multiple transformations. However, since Securisco Ruipiter developed an automated multi-module cell editing apparatus to automate genome editing in living cells, the apparatus realized cell culture, competence preparation, transformation, etc. in different modules, and transferred samples between the modules by a robot manipulation system (automated cell processing method, module, apparatus, and system including a flow-through electroporation apparatus, patent No. CN 111386334A). The device has the problems of complex system, large device volume and increased pollution risk in the sample transfer process.

The electroporation transformation is the most common transformation method with highest efficiency at present, competent cells are firstly required to be prepared before electroporation transformation is carried out, a high-speed centrifuge is used for centrifuging cells in the traditional electroporation competent cell preparation, ultrapure water is used for resuspending the cells, the cells are repeatedly washed and concentrated for many times, and the centrifuge method is difficult to realize automatic control. The invention relates to a micro-fluidic cell transformation chip, which is designed and processed, applies voltage in the process that mixture of competent cells and exogenous substances flows through a chip channel to realize cell transformation, and can continuously carry out multiple rounds of transformation after the used chip channel is automatically cleaned.

Disclosure of Invention

In view of the foregoing, the present invention provides an apparatus for automated continuous cycle cell culture and processing and methods of operating the same. The device divides the steps of cell culture, cell induction, competent cell preparation, cell electroporation transformation and the like into independent modules, the modules are sequentially connected in series to form a closed structure, the automatic operation of the modules is realized by a control program, and the device is particularly suitable for the automatic continuous circulating culture and transformation of different types of cells. The device has small pollution risk in the operation process, can realize trace cell culture, can measure the cell concentration change on line in real time, can carry out continuous multi-round cell electroporation process, and has simple operation and low cost.

To achieve the above objects, the present invention provides an apparatus for automated continuous cycle cell culture and processing and a method of operating the same, the automated apparatus comprising: the cell culture module is used for culturing cells to cell concentration or culture time data set by a user; a cell induction module for inducing cells to express certain proteins or molecules; 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 valves, pumps and pump pipes and is used for cleaning partial containers of the cell culture module, the cell induction module, the cell washing and concentrating module and the cell transformation module; 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 automatic operation of the whole device.

The cell culture module, the cell induction module, the cell washing and concentrating 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.

The cell culture module includes at least one cell culture flask for the first round of seeding with fresh cell fluid or receiving cell fluid from the cell transformation module.

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

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

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

The cell culture module also comprises a stirring device which enables the cell sap in the cell culture bottle to rotate.

In some embodiments of the present invention, the stirring device comprises a magnetic rotor, a heat dissipation fan and a magnet adhered to the fan blades of the heat dissipation fan, wherein the magnet rotates to drive the magnetic rotor in the bottle to rotate, thereby causing the cell sap to be stirred.

The cell culture module also comprises a temperature control device for controlling the temperature of the cell sap in the cell culture bottle.

In some embodiments of the invention, the temperature control device comprises a sleeve enclosing the cell culture flask, a temperature sensor and a heating resistor.

Preferably, the sleeve is made of metal material easy to conduct heat, and the inner wall of the sleeve is subjected to matte treatment.

The cell culture module further comprises an optical device for measuring the change of the cell concentration in the cell culture bottle in real time.

In some embodiments of the invention, the optical device comprises an LED and a photodiode.

Preferably, the LED has a wavelength of 600-950nm.

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.

The cell culture module further comprises a connector, a pump, a valve and a pump tube connecting the cell culture bottle with the cell induction module.

The cell culture module further comprises a connector, a pump, a valve and a pump tube for connecting the cell culture bottle with the cell transformation module.

The cell culture module further comprises a connector, a pump, a valve and a pump tube for connecting the cell culture bottle with the cleaning module.

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

The culture mode of the cell culture module comprises a time mode and an absorbance mode.

And the time mode is that the system automatically starts the next procedure after running to the set time value.

And the absorbance mode is that when the cell growth reaches a set absorbance value, the next procedure is automatically started.

The cell induction module comprises at least one culture flask for receiving cell broth from a cell culture flask of the cell culture module.

In some embodiments of the invention, the culture flask may be made of transparent glass or transparent plastic.

Preferably, the culture flask is of a glass material which is more heat conductive.

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

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

In some embodiments of the invention, the cell induction module further comprises a stirring device for rotating the cell sap in the cell culture flask.

In some embodiments of the present invention, the stirring device of the cell induction module comprises a magnetic rotor, a heat dissipation fan and a magnet adhered to the fan blades of the heat dissipation fan, wherein the magnetic rotor in the bottle is driven to rotate by the rotation of the magnet, thereby causing the cell sap to be stirred.

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

In some embodiments of the invention, the temperature control device of the cell induction module comprises a sleeve enclosing the culture bottle, a temperature sensor and a heating resistor.

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

In some embodiments of the invention, the cell induction module further comprises an optical device for determining in real time changes in cell concentration within the cell culture flask.

In some embodiments of the invention, the optical device of the cell induction module comprises an LED and a photodiode.

The cell induction module further comprises a connector, a pump, a valve and a pump tube connecting the culture flask with the cell washing and concentration module.

The cell induction module further comprises a connector, a pump, a valve and a pump tube connecting the culture bottle with the cleaning module.

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

The induction mode of the cell induction module comprises a time mode and an absorbance mode.

The cell washing and concentrating module comprises at least one conical flask for receiving cell sap from the cell inducing module culture flask, four injection pumps (a first injection pump, a second injection pump, a third injection pump and a fourth injection pump), a quick joint, a filter with a filter membrane and 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.

The cell suspension in the conical flask is pumped into the sterile ultrapure water at 4 ℃ through the filter by the first injection pump, the suspension is pumped out of the conical flask through the filter by the second injection pump, the cells are trapped on a filter membrane of the filter, sterile air is pumped into the conical flask through the filter by the third injection pump, the cells trapped on the filter membrane enter the cell suspension, and the cell suspension in the conical flask is washed and concentrated by the first injection pump and the second injection pump which sequentially and circularly operate.

The fourth syringe pump pumps the exogenous material to be transformed into the cell sap that has been washed and concentrated.

In some embodiments of the invention, the cell washing and concentration module further comprises a refrigeration plate for controlling the temperature of the conical flask.

In some embodiments of the invention, the cell washing and concentration module further comprises a stirring device for rotating the cell sap in the erlenmeyer flask.

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

In some embodiments of the invention, the cell washing and concentration module further comprises a membrane washing step. And in the membrane washing step, the first injection pump, the second injection pump and the third injection pump are operated in sequence to wash the upper filter membrane of the filter.

The cell washing and concentrating module further comprises a joint, a pump, a valve and a pump pipe which are connected with the cell transformation module.

The cell washing and concentrating module further comprises a joint, a pump, a valve and a pump pipe which are connected with the cleaning module.

In some embodiments of the invention, the cell washing and concentrating module further comprises a connector, a pump, a valve and a pump tube for discharging the liquid in the erlenmeyer flask.

The cell culture module, the cell induction module and the cell washing and concentration module are all defaulted with a waste liquid discharge process, namely, all the residual cell liquid or washing liquid in the bottle is discharged after each step of operation is finished.

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

The micro-fluidic cell conversion chip is made of materials including polymethyl methacrylate, polydimethylsiloxane, ceramics, glass or other non-conducting materials.

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

The microfluidic cell conversion chip comprises at least one fluid channel, a channel inlet and a channel outlet.

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

In some embodiments of the invention, the width of the fluid channel of the microfluidic cell conversion chip is 50 to 500 micrometers.

In some embodiments of the invention, the depth of the fluid channel of the microfluidic cell conversion chip is 50 to 200 microns.

The metal tubes are respectively inserted into the fluid channel inlet and the channel outlet of the microfluidic cell conversion chip. The metal tube is respectively connected with the high-voltage output end and the grounding end of the high-voltage amplifier.

The plastic tubes are respectively connected with the metal tubes, and the mixture containing the cells and the exogenous substances in the conical flask from the cell washing and concentrating module is pumped into the fluid channel of the microfluidic cell conversion chip through the peristaltic pump.

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

And the cells flowing out of the microfluidic cell transformation chip of the cell transformation module flow into a cell culture bottle of the cell culture module through a pump pipe to perform the next round of cell culture and treatment.

The cleaning module comprises at least one cleaning bottle, a joint for discharging liquid in the bottle, a pump, a valve and a pump pipe.

In some embodiments of the invention, the cleaning module comprises a sterilized ultrapure water cleaning bottle, a 75% ethanol cleaning bottle, and connectors, pumps, valves and pump lines connected to the cell culture bottle of the cell culture module, the culture bottle of the cell induction module, the conical bottle of the cell washing and concentration module, and the fluid channel of the microfluidic cell transformation chip of the cell transformation module.

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

In some embodiments of the 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 at least comprises an automatic mode program, a manual mode program and an absorbance (OD 600) calibration program.

The operation method of the device for automatic continuous circulation cell culture and treatment comprises the following steps:

(1) Preferably, the absorbance curve of the instrument is first calibrated. The calibration procedure is to measure the absorbance (OD 600) of freshly cultured cell sap using a commercially available spectrophotometer and to dilute the OD600 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 in sequence, each dilution having 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 readings, the instrument automatically draws a standard curve. During the cell growth process, the light intensity value is read according to the drawn curve and the instrument, and the OD600 value of the cell growth is automatically updated by the instrument every 10 seconds.

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

(3) After sterilization, the device was assembled in a clean bench and a volume of fresh cell culture solution was introduced into the culture flask of the cell culture module.

(4) The controller was automatically programmed to set the culture mode of the cell culture module, culture temperature, agitation speed, culture medium volume, transfer volume (i.e., the volume of cell broth transferred to the next unit), and wash solution volume.

(5) The induction mode, induction temperature, stirring speed, transfer volume (i.e., the volume of cell fluid transferred to the next unit), and washing fluid volume of the cell induction module are set.

(6) The operating temperature, the stirring speed, the pumping volume, the pumping speed, the discharging speed and the washing liquid volume of the cell washing and concentrating module are set.

(7) And setting the high-pressure period and the high-pressure duty ratio of the cell transformation module and the running speed of the pump.

(8) And setting the cycle number, and sending an instruction to start the device.

Due to the adoption of the technical scheme, the invention has the following advantages: the device completely automates the whole process of cell culture, cell induction, competent cell preparation, cell transformation and cell resuscitation, and the whole process can be continuously operated in a multi-cycle manner; compared with a large device with a mechanical arm, the device is more miniaturized and simpler to operate; compared with the traditional electric rotating cup conversion mode, the microfluidic chip of the device can realize cell flow electric conversion continuously in multiple rounds and has higher conversion efficiency.

Drawings

Fig. 1 is a schematic view of the external overall structural design of the automation system of the invention.

FIG. 2a is a schematic design diagram of a cell culture module 1 according to an embodiment of the present invention.

FIG. 2b is a schematic diagram of the position of the optical elements of the cell culture module 1 for measuring cell growth according to the embodiment of the present invention.

FIG. 2c is a graph of the calibration of the measured light intensity of the cell culture module 1 to the measured OD600 of the spectrophotometer provided by the embodiment of the present invention.

FIG. 2d is a graph of the growth curve of E.coli cells in a custom cell culture flask as determined by cell culture module 1 provided in an example of the present invention, as compared to the growth of a 100mL shake flask.

FIG. 2e is a plot of E.coli growth over the first 0-3 hours of FIG. 2 d.

FIG. 3a is a schematic diagram of a cell induction module 2 according to an embodiment of the present invention.

FIG. 3b is a diagram of the example of the present invention, which provides the case that the E.coli cells grown in the cell culture module 1 to OD600=0.4 are cultured and induced at 42 ℃ of the cell induction module 2 for different time periods.

FIG. 3c shows the efficiency of homologous recombination of E.coli cells induced at 42 ℃ for different times in the cell induction module 2 of example 3 of the present invention.

FIG. 4a is a schematic structural design diagram of the cell washing and concentration module 3 according to the embodiment of the present invention.

FIG. 4b shows the competent cell volume and the residual cell amount prepared by the continuous treatment of 8 cycles of the single filter of the cell washing and concentrating module 3 provided in example 4 of the present invention.

FIG. 5a is a schematic diagram of cell flow electroporation transfer of the microfluidic cell transfer chip 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 is 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 a graph showing 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 diagram of a cleaning module 5 according to an 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. a cell culture module; 2. a cell induction module; 3. a competence preparation module; 4. a cell transformation module; 5. a cleaning module; 6. a control module; 10. a culture medium bottle; 101. a culture medium; 11. a culture bottle; 121. a sleeve; 122. a circuit board; 131. a temperature sensor; 132. a heating resistor; 141. an LED; 142. a photodiode; 151. a magnetic rotor; 152. a heat-dissipating fan; 153. a magnet; 21. a culture bottle; 221. a sleeve; 222. a circuit board; 231. a temperature sensor; 232. a heating resistor; 241. an LED; 242. a photodiode; 251. a magnetic rotor; 252. a heat radiation fan; 253. a magnet; 31. a conical flask; 321. a first syringe pump; 322. a second syringe pump; 323. a third syringe pump; 324. a fourth syringe pump; 33. a filter; 34. a refrigeration plate; 351. a magnet; 352. a magnetic rotor; 353. a heat radiation fan; 40. a peristaltic pump; 41. a microfluidic electroporation conversion chip; 42. a high voltage amplifier; 43. a stainless steel tube; 44. a silicone tube; 45. a cell and a nucleic acid mixture; 51. ultrapure water; 52. 75% ethanol; 53. a pump and a pump tube; 61. a controller; 62. and a control circuit board.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

Referring to fig. 1 in combination, the present embodiment provides an apparatus for automated continuous cycle cell culture and processing, comprising: the cell culture module 1 is used for culturing cells to grow to cell concentration or culture time data set by a user; a cell induction module 2 for inducing cells to express certain proteins or molecules; a cell washing and concentrating module 3 for preparing electroporation transformation competent cells; a cell transformation module 4 for electroporation transforming competent cells; a cleaning module 5 for cleaning part of the containers of the cell culture module, the cell induction module, the cell washing and concentration module and the cell transformation module; and the control module 6 is used for controlling the whole program to run.

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 pipe to form a closed loop.

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

The cell culture module 1 further comprises a temperature control device for controlling the temperature of the cell sap in the cell culture bottle. The temperature control device comprises 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 a metal material easy to conduct heat, and the inner wall of the sleeve is subjected to matte treatment.

The cell culture module 1 further comprises a circuit board 122 for receiving commands sent by the control module 6, monitoring data from the cell culture flask 11 and sending commands to the control module 6.

The cell culture module 1 further comprises a stirring device for rotating the cell sap in the cell culture bottle. The stirring means includes a magnetic rotor 151, a heat-radiating fan 152, and a magnet 153 adhered to the fan blades of the heat-radiating fan 153..

The cell culture module 1 further comprises an optical device for measuring the change in cell concentration in the cell culture flask in real time. Specifically, the optical device includes an LED141 and a photodiode 142. The wavelength of the LED141 is 600-950nm.

The cell culture module 1 further comprises at least one medium bottle 10 for holding medium 101 and a joint, a peristaltic pump, a one-way valve and a pump tube for connecting the medium bottle to the cell culture bottle 11.

The cell culture module 1 further comprises a joint, a peristaltic pump, a one-way valve and a pump pipe which are used for connecting the cell culture bottle 11 with the cell induction module 2; the device also comprises a joint, a peristaltic pump, a one-way valve and a pump pipe which are used for connecting the cell culture bottle 11 with the cell transformation module 4; the device also comprises a joint, a peristaltic pump, a one-way valve and a pump pipe which are used for connecting the cell culture bottle 11 with the cleaning module 5; the device also comprises a joint, a peristaltic pump, a one-way valve and a pump pipe for discharging liquid in the cell culture bottle 11.

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

And 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.

The cell induction module 2 comprises at least one culture flask 21 for receiving cell broth from the cell culture flask 11. The culture flask 21 may be made of transparent glass or transparent plastic. Preferably, the culture flask 21 is made of glass material which is more heat conductive, and the shape of the culture flask can be round, square or polygonal.

The cell induction module 2 also includes a circuit board 222 that receives commands from the control module 6, monitors flask 21 data, and sends commands to the control module.

The cell induction module 2 further comprises a stirring device for rotating the cell fluid in the culture flask 21. Specifically, the stirring device includes a magnetic rotor 252, a heat radiating fan 253, and a magnet 251 adhered to the blades of the heat radiating fan 253.

The cell induction module 2 further comprises 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 enclosing the culture bottle 21, a temperature sensor 231, and a heating resistor 232. Preferably, the sleeve 221 is made of a metal material with easy heat conduction.

The cell induction module 2 further comprises an optical device for measuring the change in cell concentration in the culture flask 21 in real time. Specifically, the optical device includes an LED241 and a photodiode 242.

The cell induction module 2 further comprises a joint, a peristaltic pump, a one-way valve and a pump pipe which are used for connecting the culture bottle 21 with the cell washing and concentrating module 3; the device also comprises a joint, a peristaltic pump, a one-way valve and a pump pipe which are used for connecting the culture bottle 21 with the cleaning module 3; the device also comprises a connector for discharging liquid in the culture bottle 21, a peristaltic pump, a one-way valve and a pump pipe.

The induction mode of the cell induction module 2 includes a time mode and an absorbance mode.

The cell washing and concentration module 3 comprises a conical flask 31 for receiving cell fluid from a culture flask 21, four syringe pumps (i.e. a first syringe pump 321, a second syringe pump 322, a third syringe pump 323 and a fourth syringe pump 324), a four-way joint 325 and a filter 33 with a filter membrane.

The four-way joint 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 injection pump 321 pumps sterile ultrapure water at 4 ℃ into the cell suspension in the conical flask 31 through the filter 33, the second injection pump 322 pumps the suspension out of the conical flask 31 through the filter 33, the cells are trapped on a filter membrane of the filter 33, the third injection pump 323 pumps sterile air into the conical flask 31 through the filter 33, the cells trapped on the filter membrane enter the cell suspension, and the first injection pump 321 and the second injection pump 322 are sequentially circulated to wash and concentrate the cell sap in the conical flask 31.

The fourth syringe pump 324 pumps the exogenous material to be transformed into the cell sap that has been washed and concentrated.

The cell washing and concentrating module 3 further comprises a refrigerating sheet 34 for controlling the temperature of the conical flask 31.

The cell washing and concentrating module 3 further comprises a stirring device for rotating the cell sap in the erlenmeyer flask 31. Specifically, the stirring device includes a magnetic rotor 352, a heat radiating fan 353, and a magnet 351 attached to a fan blade of the heat radiating fan 353.

The cell washing and concentration module 3 further comprises a membrane washing step. The membrane washing step circularly operates the upper filtration membrane of the washing filter 33 in sequence by using a first injection pump 321, a second injection pump 322 and a third injection pump 323.

The cell washing and concentrating module 3 further comprises a joint for discharging the liquid in the conical 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, namely, after the operation of the modules is finished, residual cell liquid or washing liquid in the cell culture bottle 11 of the cell culture module 1, the culture bottle 21 of the cell induction module 2 and the conical flask 31 of the cell washing and concentration module 3 is completely discharged.

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

The micro-fluidic cell conversion chip 41 is made of materials including polymethyl methacrylate, polydimethylsiloxane, ceramic, glass or other non-conducting materials. The microfluidic cell transformation chip 41 may be subjected to one or more sterilization methods, including high temperature sterilization, ultraviolet sterilization, and ethanol sterilization. The microfluidic cell conversion 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 conversion chip 41 can be adjusted according to different cell types.

The metal tubes 43 are inserted into the fluid passage inlet 412 and the passage outlet 413, respectively. The metal tube 43 is connected to the high voltage output terminal and the ground terminal of the high voltage amplifier 42, respectively.

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

The high-pressure amplifier 42 is turned on before the peristaltic pump controlling the mixture of cells and exogenous materials enters the chip channel 411 is operated, and the high-pressure amplifier 42 is turned off after all the cell sap flowing out of the fluid channel 411 enters the cell culture flask 11 of the cell culture module 1.

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, and then the next round of cell culture and treatment is performed.

The cleaning module 5 comprises a cleaning bottle 51 containing 75% ethanol, a cleaning bottle 52 containing sterilized ultrapure water, and a connector, a peristaltic pump, a one-way valve and a pump pipe for connecting the cleaning bottles 51 and 52 with the cell culture bottle 11 of the cell culture module 1, the culture bottle 21 of the cell induction module 2, the conical bottle 31 of the cell cleaning and concentration module 3 and the fluid channel 411 of the microfluidic cell conversion chip 41 of the cell conversion module 4.

The cleaning module 5 is operated to sequentially use the cleaning liquids in the cleaning bottle 51 and the cleaning bottle 52 to clean the module containers after the operation of each module connected with the cleaning module is finished, that is, after the operation of the cell culture module 1 is finished, the 75% ethanol in the cleaning bottle 51 and the ultrapure water in the cleaning bottle 52 are used to sequentially clean the cell culture bottle 11 of the cell culture module 1, and the culture bottle 21 of the cell induction module 2, the conical flask 31 of the cell cleaning and concentration module 3 and the microfluidic cell conversion chip 41 of the cell conversion module 4 are also cleaned in the same manner.

The control module 6 comprises a controller 61 and a control circuit 62. The controller 61 comprises at least an automatic mode program, a manual mode program, and an absorbance (OD 600) 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 for sending a command input by a user in advance to each module and the peristaltic pump.

Example 2

The cell culture module 1 for the automatic continuous circulation cell culture and processing device provided by the invention is used for realizing the automatic culture of escherichia coli, and fig. 2a is a design schematic diagram of the cell culture module 1. The cell culture module 1 of the present embodiment uses a customized flask as the culture flask 11, the inner side of which has a length of 19mm, an inner width of 12mm, and a volume of 10mL. FIG. 2b shows the optical element mounting position when cells are cultured using a square flask.

The absorbance (OD 600) of freshly cultured E.coli cell sap was measured using a commercial spectrophotometer, and the OD600 values of the cell sap were diluted to 0,0.1,0.2,0.4,0.6,0.8,1,1.2,1.4,1.6,1.8,2 in that order, with a volume of not less than 3mL per dilution. The instrument absorbance (OD 600) calibration procedure was opened and the dilutions were transferred to cell culture flasks 11 in sequence, and the light intensity values read by the instrument were recorded within the procedure. After all readings, the instrument automatically draws a standard curve. FIG. 2c is a graph showing the light intensity of E.coli measured at different concentrations in the cell culture module 1 calibrated to OD600 measured in a spectrophotometer. During the culture of the Escherichia coli cells, the OD600 value of the growth of the Escherichia coli cells is automatically updated by the instrument every 10 seconds according to the drawn curve and the reading light intensity value of the instrument. 100. Mu.l of fresh cell culture medium with OD600=3 was added to the culture flask 11 of the cell culture module 1, and the volume of the medium 101 was 3mL and the culture temperature was 30 ℃. FIG. 2d is a comparison of E.coli growth in a custom cell culture flask 11 with a 100mL shake flask. FIG. 2e shows cell growth up to the first 0-3 hours of FIG. 2 d.

Example 3

The cell induction module 2 for the automatic continuous circulation cell culture and treatment device provided by the invention is used for realizing the automatic induction of cells. FIG. 3a shows a schematic design of the cell induction module 2 provided in this embodiment. Coli ATCC 8739 (port mage) cells of example 2 were cultured to 600=0.4, and 2mL of cell solution was transferred to the culture flask 21 of the cell induction module 2 by an automatic syringe pump, and the culture flask 21 of this example was a 20mL screw-top black cap flask. The cell fluid was cultured at 42 ℃ for various periods of time to induce recombinase expression. Wherein the plasmid pORTMAGE is derived from reference Nyes et al, proc Natl Acad Sci USA.2016,113 (9): 2502-7.

1mL of cell sap is taken for centrifugal washing to prepare competent cells, artificially synthesized ssDNA which can lead the expression of the gene lacZ to be stopped in advance is electrically transformed into the competent cells, the recovered cells are coated with LB solid plates containing IPTG (isopropylthio-beta-D-galactoside) and X-gal (5-bromo-4-chloro-3-indole-beta-D-galactoside), and when single colonies grow out, the number of white colonies is counted. FIG. 3b shows the growth of cells cultured at 42 ℃ for various periods of time. FIG. 3c shows the proportion of white colonies, i.e., the efficiency of homologous recombination, after induction culture at 42 ℃ for various periods of time.

Example 4

The cell washing and concentrating module 3 for the automatic continuous circulating cell culture and treatment device provided by the invention is used for realizing automatic competent cell preparation. After culturing the cell fluid of example 3 at 42 ℃ for 15min, 1mL of the cell fluid was transferred to the conical flask 31 of the cell washing and concentration module 3. FIG. 4a is a schematic diagram of the structure of a cell washing and concentrating module 3 according to an embodiment of the present invention.

The four injection pumps are all multi-channel valve head continuous injection pumps. One valve head of the first injection pump 321 is connected with ultrapure water, and the other valve head is connected with a four-way joint 325; one valve head of the second injection pump 322 is connected with the four-way joint 325, and the other valve head is connected with the waste liquid container; one valve head of the third injection pump 323 is connected with the 0.2 mu m filter 33 with a filter membrane, and the other valve head is connected with the four-way joint 325; one valve head of the fourth injection pump 324 is connected with the ssDNA storage conical flask, and the other valve head is connected with the conical culture flask 31; setting the liquid inlet flow rate of a first injection pump 321 to be 5s/mL, the liquid discharge flow rate to be 10s/mL, the volume of ultrapure water to be 1mL, the liquid inlet flow rate of a pump 322 to be 30s/mL, the liquid discharge flow rate to be 5s/mL, the liquid discharge volume to be 1.15mL, circulating operation of the pump 321 and the pump 322 for 10 times, the air inlet flow rate of a pump 323 to be 5s/mL, the air exhaust flow rate to be 10s/mL, the volume of sterile air to be 2mL, the liquid inlet flow rate of a pump 324 to be 15s/mL, the liquid discharge flow rate to be 15s/mL and the ssDNA volume to be 0.05mL.

After each run, the remaining cell volume and cell mass were recorded. In addition, each run is followed by a membrane washing step, i.e., the first syringe pump 321 and the third syringe pump 323 are cyclically run for 3 times, and then the next competent preparation process is entered.

Each filter was run 8 times. FIG. 4b shows the cell wash and concentration module 3 single filter continuous treatment for 8 cycles to prepare competent volume and remaining cell mass.

Example 5

The cell transformation is realized by using the cell transformation module 4 for the automatic continuous circulating cell culture and treatment device provided by the invention. In the present invention, the microfluidic chip-based cell flow electroporation transformation is used instead of the laboratory electroporation using an electric rotating cup, wherein the microfluidic cell transformation chip 41 is shown in FIG. 5 a. 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 micrometers, the length from the inlet to the outlet of the chip is 5800 micrometers, and the chip channel structure is shown in fig. 5 b. The competent cells prepared in example 4 were injected into microfluidic cell transformation chips with different depths and widths at a rate of 200 μ L/min, respectively, and compared with an electric rotor method, and the results are shown in fig. 5c and 5d, where the transformation efficiency was the highest when both the channel width and the channel depth were 100 μm, and both were superior to the control of the electric rotor method.

Example 6

The flow of the operation method of the device for automatic continuous circulation cell culture and treatment according to the above embodiment of the present invention is as follows:

1) The absorbance curve of the instrument was first calibrated. The specific calibration procedure is to measure the absorbance (OD 600) of freshly cultured cell sap by using a commercial spectrophotometer, and to dilute the OD600 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 in sequence, wherein the volume of each diluted liquid is not less than 3mL. The absorbance (OD 600) calibration procedure was opened and the dilutions were transferred to cell culture flasks 11 in sequence, and the light intensity values read by the instrument were recorded in the procedure. After all readings, 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.

2) The cell culture bottle 11 of the cell culture module 1, the culture bottle 21 of the cell induction module 2, the cleaning bottle 51 of the cleaning module 5 containing sterilized ultrapure water, and the pump tube of the apparatus were sterilized at high temperature, and the conical bottle 31 of the cell washing and concentration module 3, the microfluidic cell conversion chip 41 of the cell conversion module 4, and the connection pump tube were sterilized with 75% ethanol.

3) After sterilization, the entire apparatus is assembled in a sterile clean bench and a volume of fresh cell culture solution is introduced into the cell culture flask 11 of the cell culture module 1.

4) The automatic mode program of the controller 61 is turned on, and the culture mode, culture temperature, culture medium volume, transfer volume (i.e., volume for transferring cell sap to the next unit), operation volume of the cleaning solution 75% ethanol for cleaning bottle 51, and operation volume of the ultra-pure water for cleaning solution for cleaning bottle 52 of the cell culture module 1 are set.

5) The induction mode, induction temperature, transfer volume (i.e., volume for transferring cell sap to the next unit), 75% ethanol per run volume for washing the bottle 51, and ultrapure water per run volume for washing the bottle 52 of the cell induction module 2 were set.

6) The operation temperature of the cell washing and concentration module 3, the pumping volume, the pumping speed, and the discharging speed of the first syringe pump 321, the second syringe pump 322, the third syringe pump 323, and the fourth syringe pump 324 are set.

7) The high pressure period and high pressure duty ratio of the cell transformation module 4 and the operation speed of the peristaltic pump 40 are set.

8) And setting cycle times and sending 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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