CN103074213A - Living cell detection device and detection method for tissue engineering reactor - Google Patents
Living cell detection device and detection method for tissue engineering reactor Download PDFInfo
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- CN103074213A CN103074213A CN2012105791503A CN201210579150A CN103074213A CN 103074213 A CN103074213 A CN 103074213A CN 2012105791503 A CN2012105791503 A CN 2012105791503A CN 201210579150 A CN201210579150 A CN 201210579150A CN 103074213 A CN103074213 A CN 103074213A
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Abstract
The invention provides a living cell detection device and a detection method for a tissue engineering reactor, which can detect the cell growth condition of tissue culture objects in the tissue engineering biological reactor under the preconditions of not damaging the cell activity and functions and not interrupting the cultivation process. The living cell detection device according to the embodiment of the invention comprises a flushing loop and a detection loop, wherein the flushing loop and the detection loop are in parallel connection with a culture loop of the tissue engineering reactor, and are combined with a pipe through a three-way valve; and the conversion of the loops is controlled through opening or closing a valve. The flushing loop comprises a flushing liquid storage bottle 101, a liquid driving pump 103 and a waste liquid bottle 109, and is used for flushing of tissue culture objects 111 before and after the detection; and the detection loop comprises a detection liquid storage bottle 201, the liquid driving pump 103, a detector 203, a computer 204 and the waste liquid bottle 109, and is used for detecting living cells on the tissue culture objects 111.
Description
Technical field
The present invention relates to cell cultures, field of tissue engineering technology, relate more specifically to a kind of proofing unit and detection method that can detect viable cell growing state on tissue culture in the organizational project bio-reactor or the anatomic implants, detect the tissue culture procedures cell in the truth of some specific tissue engineering brackets or anatomic implants growth and survival for online or off-line continuous and quantitative, the influence factor of Growth of Cells and survival is carried out quality control to sample in the assessment tissue culture.
Background technology
An important application of tissue engineering technique is exactly the external structure anatomic implants, seed cell with vitro culture with Method of Tissue Engineering external structure anatomic implants namely, be inoculated on the tissue engineering bracket of natural or synthetic, after vitro culture, be transplanted to again in the body, to substitute the tissue of disease damage.Unprecedented chance has been created in the treatment that develops into congenital disorders and acquired disease of tissue engineering technique.
Amount of viable cell on the anatomic implants is a very important parameter, and it is the basis of analysis of cells growth, survival and metabolism, is the primary parameter of control tissue culture procedures, also is the important indicator whether the assessment anatomic implants has physiological function.In the culturing process of anatomic implants, often need to understand in time, quantitatively the growing state of cell, in order to culturing process is monitored.
But present tissue engineering reactor lack can the tissue culture in cultivating be carried out online, the device of continuous, detection by quantitative, in tissue culture, in most cases can only carry out discrete, non-dynamic end point determination to the growing state of cell on anatomic implants, and can not carry out continuous, the dynamic detection.If Continuous Observation Growth of Cells and survival condition need to be cultivated a large amount of samples simultaneously, in culturing process, take out culture and carry out end point determination.This can cause a large amount of wastes of support, seed cell, substratum and culture device.The tissue culture procedures rare for seed cell, that culturing process is complicated, cultivation is difficult is subject to the restriction of the scale of cultivating, and the continuous detecting of Growth of Cells and survival and assessment just are difficult to realization more in the culturing process.Therefore, field of tissue engineering technology in the urgent need to set up Simple fast, can be in not damaging cells activity and physiological function, do not interrupt carrying out the quantitative proofing unit of viable cell and detection method under the prerequisite of culturing process.
Chinese patent application (200680041273.4) discloses a kind of apparatus and method for detection of activity of living cells, uses the imaging sensor analysis of cells at the post-stimulatory fluoroscopic image of fluorescence dye, obtains the detection data of cytoactive.
The object of the present invention is to provide a kind of can be online or device and the corresponding detection method of cytoactive and growing state on the anatomic implants of cultivating in off-line, continuous, the quantitative detection tissue engineering reactor.Compared with prior art, the invention has the advantages that this device has detection loop and a flush loop that is in parallel with the tissue culture loop that is in parallel with the tissue culture loop, can detect cytoactive and Growth of Cells situation in the situation that do not interrupt culturing process, and can in time remove detection reagent to the harm of cell; Another advantage is the simple effectively easily enforcements of these apparatus and method, and is also not high to the requirement of tissue culturing equipment and test set.Its implementation result be to realize Growth of Cells in the tissue culture procedures controlledization, can monitoring, be conducive to for anatomic implants clinical application, formulate relevant GLP and GMP standard foundation be provided.
Summary of the invention
According to an aspect of the present invention, provide a kind of can be online or the device of cytoactive and growing state on the anatomic implants of cultivating in off-line, continuous, the quantitative detection tissue engineering reactor, it is characterized in that comprising: a detection loop in parallel with the cultivation loop of tissue engineering reactor and a flush loop in parallel with the cultivation loop of tissue engineering reactor.Detect loop and flush loop and combine by valve and pipeline, and detect the switching of loop and flush loop by valve opening and closing control.
The described detection loop in parallel with the cultivation loop of tissue engineering reactor comprises that one is detected the liquid liquid storage bottle, links to each other with the tissue culture chamber by pipeline; A liquid driving pump is arranged on and detects between liquid liquid storage bottle and the tissue culture chamber, is used for driving liquid and flows in the loop; A detector is arranged on downstream, tissue culture chamber, for detection of signal intensity; Constant flow pump driving detection liquid circulates in the loop of this sealing in testing process.
The described flush loop in parallel with the cultivation loop of tissue engineering reactor comprises a washing fluid liquid storage bottle, links to each other with tissue culture chamber, detector successively by pipeline; A waste liquid bottle that is arranged on the detector downstream, a constant flow pump is arranged between washing fluid liquid storage bottle and the tissue culture chamber, be used for to drive liquid from washing fluid liquid storage bottle flow through successively tissue culture chamber and detector, flow into waste liquid bottle, finish detecting the flushing in loop;
An another aspect of the present invention provides the method for cytoactive and growing state on the anatomic implants of cultivating in a kind of can be online, continuous, quantitative detection tissue engineering reactor, it is characterized in that comprising:
1, closes tissue culture loop upstream and downstream
2, switch to flush loop;
3, flushing tissue culture;
4, close flush loop, the open detection loop;
5, detection reagent is injected in the tissue culture chamber;
6, allow detection reagent circulate in detecting the loop, the optical density(OD) of detection original state or fluorescence intensity are as initial value (reference);
7, allow detection reagent in detecting the loop, circulate, allow detection reagent react to specific time;
Optical density(OD) or fluorescence intensity when 8, detection reaction finishes with signal and the reference contrast that detects, are calculated amount of viable cell;
9, switch to flush loop
10, flushing tissue culture;
11, close flush loop, switch to and cultivate the loop, continue tissue culture.
In one embodiment, described reaction reagent can be that the reagent of or low toxicity nontoxic to cell comprises: the reagent such as the Alamar Blue that contain resazurin (Resazurin) dyestuff, the reagent that contains diacetyl fluorescein (FDA) dyestuff, the reagent that contains serum lactic dehydrogenase, the reagent that contain fluorine derivative (fuo derivative), furan derivatives (fura derivative), indole derivatives (indo derivate), rhod-2, calcium is green and quin-2 forms.
Cell on described tissue culture 111 in the embodiment comprises: the various histocytes of former culture, the clone of cultured continuously, have the stem cell of multiple differentiation potential or single differentiation potential.These Growth of Cells are on the tissue engineering bracket or on the microcarrier.Described tissue culture 111 can be to be fixed in the tissue culture chamber of tissue engineering reactor, also can be that suspended state is cultivated in the tissue culture chamber.
Description of drawings
Fig. 1 has illustrated to show the structure of flush loop in the viable cell proofing unit according to an embodiment of the invention;
Fig. 2 has illustrated to show the structure that detects the loop in the viable cell proofing unit according to an embodiment of the invention;
Fig. 3 has illustrated to show the structure of the viable cell proofing unit for tissue engineering reactor according to an embodiment of the invention, wherein flush loop and detect the loop on a path;
Fig. 4 has illustrated to show the switching of structure and the path direction of T-valve according to an embodiment of the invention;
Fig. 5 has illustrated to show the structure in the tissue culture chamber with the cell retention device according to an embodiment of the invention;
Fig. 6 has shown the schema that viable cell detects that carries out according to an embodiment of the invention.
Embodiment
As shown in Figure 1, a kind of viable cell proofing unit for tissue engineering reactor according to an embodiment of the invention comprises that a flush loop in parallel with the tissue culture loop comprises: a washing fluid liquid storage bottle 101, a T-valve 102, a liquid driving pump 103, a T-valve 104, a tissue culture chamber 105, tissue culture 111, a T-valve 106, a test chamber 107, a T-valve 108, a waste liquid bottle 109, pipeline 110 connects into a loop to above-mentioned parts.Be used for flushing tissue culture 111 and detection loop before detecting, remove the nutrient solution that affects detection reaction; Or after detecting amount of viable cell, wash tissue culture 111 and detect the loop, remove and detect liquid, eliminate and detect the liquid long-term existence to the impact of cell.
Described tissue culture 111 is positioned at tissue culture chamber 105, can be to be fixed on the 105 interior cultivations of tissue culture chamber, also can be to be suspended in the 105 interior cultivations of tissue culture chamber.
Described T-valve is used for the switching of control stream or stream assembly, thereby finishes the switching of stream.Wherein said T- valve 104 and 106 lays respectively at the upstream and downstream in tissue culture chamber 105, finishes the switching of stream between tissue culture loop 112 and detection or flush loop; T-valve 102 is positioned at washing fluid liquid storage bottle upstream, is used for the switching of control washing fluid liquid storage bottle.
Described liquid driving pump 103 is arranged between washing fluid liquid storage bottle 101 and the tissue culture chamber 105, be used for to drive liquid from washing fluid liquid storage bottle 101 flow through successively tissue culture chamber 105 and test chamber 107, flow into waste liquid bottle 109, finish the flushing to tissue culture.
In the specific implementation process of flushing:
(1) swivel tee valve 102 is unimpeded to stream between washing fluid liquid storage bottle 101 and the liquid driving pump 103; Swivel tee valve 104 is unimpeded to stream between liquid driving pump 103, the tissue culture chamber 105, and stream is closed between 112 upstreams, tissue culture loop and the tissue culture chamber 105; Swivel tee valve 106 is unimpeded to stream between tissue culture chamber 105 and the test chamber 107, and stream is closed between 112 downstreams, 105 tissue culture loops, tissue culture chamber; Swivel tee valve 108 is unimpeded to stream between test chamber 107 and the waste liquid bottle 109.
(2) open liquid driving pump 103, make washing fluid replace gradually other liquid of stream, until washing fluid is full of stream fully, liquid flows into waste liquid bottle 109 in this process.
As shown in Figure 2, a kind of viable cell proofing unit for tissue engineering reactor according to an embodiment of the invention comprises that a detection loop in parallel with the tissue culture loop comprises: one is detected liquid liquid storage bottle 201, a T-valve 202, a liquid driving pump 103, a T-valve 104, a tissue culture chamber 105, tissue culture 111, a T-valve 106, a detector 203, the test chamber 107 that is connected with signal piping in the detector, 108, one waste liquid bottles 109 of a T-valve, pipeline 110 connects into a loop to above-mentioned parts, detector is connected with a computer 204, by computer 204 control detectors and detect processing and the output of data.T-valve 202 is detecting between liquid liquid storage bottle 201 and the liquid driving pump 103, finishes stream and is detecting the loop, detecting the switching between circulation loop or the flush loop; T- valve 104 and 106 lays respectively at the upstream and downstream in tissue culture chamber 105, finishes the switching of stream between tissue culture loop and detection or flush loop; T-valve 108 is finished stream in the switching that detects between loop or the detection circulation loop between waste liquid bottle 109 and detection liquid liquid storage bottle 101.
In the specific implementation process that detects:
(1) swivel tee valve 202 is unimpeded to stream between detection liquid liquid storage bottle 201 and the liquid driving pump 103, stream is closed between detection liquid liquid storage bottle 201 and the waste liquid bottle 109, swivel tee valve 104 is unimpeded to stream between liquid driving pump 103, the tissue culture chamber 105, and stream is closed between 112 upstreams, tissue culture loop and the tissue culture chamber 105; Swivel tee valve 106 is unimpeded to stream between tissue culture chamber 105 and the test chamber 107, and stream is closed between 112 downstreams, 105 tissue culture loops, tissue culture chamber; Swivel tee valve 108 is unimpeded to stream between test chamber 107 and the waste liquid bottle 109, and stream is closed between waste liquid bottle 109 and the detection liquid liquid storage bottle 201.
(2) open liquid driving pump 103, make and detect other liquid that liquid is replaced stream gradually, be full of stream fully until detect liquid, liquid flows into waste liquid bottle 109 in this process.
(3) swivel tee valve 202 cuts out to detecting liquid liquid storage bottle 201, and stream is unimpeded between liquid driving pump 103 and detection liquid liquid storage bottle 201 downstreams; Swivel tee valve 108 to waste liquid bottle 109 is closed, stream is unimpeded between liquid driving pump 103 and the test chamber 107, thereby form the loop of a sealing, detecting liquid is driven by liquid driving pump 103, in tissue culture chamber 105 and test chamber 107, circulate, so that fully react at the detection agent of the reaction times internal volume of stipulating and the cell on the anatomic implants; Detect the effect of playing mass transfer and mixing that circulates of liquid, make that to detect the liquid reaction more abundant, improve the signal of reaction, prevent that nutrition from exhausting, metabolic waste transports.
Wherein said pipeline 110 is comprised of flexible pipe or hard tube nontoxic, that can tolerate moist heat sterilization or chemosterilization such as oxyethane, alcohol sterilization, the diameter of pipe at 0.1mm between the 3mm.
Wherein said test chamber 107 is the cavitys that can carry out fluorescence, absorbancy or chemiluminescence detection; Detector 110 is the EM equipment module that can carry out fluorescence, absorbancy or chemiluminescence detection; Detector is connected with a computer 112, controls detectors by computer 112, and detects processing and the output of data.
The reagent that wherein said detection liquid can be or low toxicity nontoxic to cell comprises: the reagent such as the Alamar Blue that contain resazurin (Resazurin) dyestuff, the reagent that contains the diacetyl fluorescein(e) dye, the reagent that contains serum lactic dehydrogenase, the reagent that contain fluorine derivative (fluo derivative), furan derivatives (fura derivative), indole derivatives (indo derivate), rhod-2, calcium is green and quin-2 forms.And detect liquid and contain corresponding essential nutritive substance and the pH environment of histocyte maintenance metabolism, such as the cell culture fluid consistent with cell culture condition to be measured or D-Hanks liquid, PBS liquid etc., and these materials do not affect the carrying out of detection reaction and the detection of signal.
As shown in Figure 3, a kind of viable cell proofing unit for tissue engineering reactor according to an embodiment of the invention, wherein flush loop and detection loop are incorporated on a path, comprise: a washing fluid liquid storage bottle 101, a T-valve 102, one is detected liquid liquid storage bottle 201,103, one T-valve 104 of 202, one liquid driving pumps of a T-valve, a tissue culture chamber 105,106, one detectors 203 of 111, one T-valve of tissue culture, the test chamber 107 that is connected with signal piping in the detector, 108, one waste liquid bottles 109 of a T-valve, pipeline 110 connects into a loop to above-mentioned parts, detector is connected with a computer 204, by computer 204 control detectors and detect processing and the output of data.
Described detection liquid liquid storage bottle 201, washing fluid liquid storage bottle 101, waste liquid bottle 109 are set in turn in the upstream of liquid driving pump 103, link to each other with the pipeline of liquid driving pump 103 upstreams by T-valve 202,102,108 respectively, detect the switching of loop and flush loop by the open and close controlling of T-valve.
Described flush loop and detect the loop combination mode and can simplify the viable cell proofing unit, in the viable cell testing process by the stream easy switching finish flushing and the detection of sample.
As shown in Figure 4, T-valve according to an embodiment of the invention, by the rotation of T-valve inner spool, the control liquid flow path is unimpeded on two or three directions, thereby finishes the switching of stream.
It should be noted that also can be used in two two-way valves are set on the loop replace described T-valve, as long as it can finish the process that corresponding stream switches.
As shown in Figure 5, tissue culture according to an embodiment of the invention chamber 105, a cell retention device 501 is arranged in its exit, when the anatomic implants suspension growth was in tissue engineering reactor, cell retention device 501 makes the liquid such as washing fluid, detection liquid, and cell was trapped within the culture chamber by 105 inflow detected downstream chambeies 107, tissue culture chamber.Described cell retention device can be core, filter or screen cloth, and it holds back the aperture at 1-10 μ m.
As shown in Figure 6, the schema that carries out the viable cell detection according to an embodiment of the invention.In an implementation process in conjunction with the concrete detection of Fig. 3 device:
(1) swivel tee valve 104 cuts out to stream between 112 upstreams, tissue culture loop and the tissue culture chamber 105, and stream is unimpeded between liquid driving pump 103, the tissue culture chamber 105; Swivel tee valve 106 cuts out to stream between 112 downstreams, 105 tissue culture loops, tissue culture chamber, and stream is unimpeded between tissue culture chamber 105 and the test chamber 107; Thereby close the tissue culture loop.
(2) swivel tee valve 102 is unimpeded to stream between washing fluid liquid storage bottle 101 and the liquid driving pump 103; Swivel tee valve 202 cuts out to detecting liquid liquid storage bottle 201, and stream is unimpeded between washing fluid liquid storage bottle 101 and the liquid driving pump 103; Swivel tee valve 108 is unimpeded to stream between test chamber 107 and the waste liquid bottle 109, and stream is closed between waste liquid bottle 109 and the washing fluid liquid storage bottle 201; Thereby make flush loop unimpeded.
(3) open liquid driving pump 103, make washing fluid replace gradually other liquid of stream, until washing fluid is full of stream fully, liquid flows into waste liquid bottle 109 in this process, thereby finishes the flushing of tissue culture.
(4) swivel tee valve 202 is unimpeded to stream between detection liquid liquid storage bottle 201 and the liquid driving pump 103, and stream is closed between detection liquid liquid storage bottle 201 and the washing fluid liquid storage bottle 101; Swivel tee valve 108 is unimpeded to stream between test chamber 107 and the waste liquid bottle 109, and stream is unimpeded between waste liquid bottle 109 and the washing fluid liquid storage bottle 101; Swivel tee valve 102 cuts out washing fluid liquid storage bottle 101, and stream is unimpeded between washing fluid liquid storage bottle 101 and the liquid driving pump 103; Thereby finish flush loop to the switching that detects the loop.
(5) open liquid driving pump 103, make and detect other liquid that liquid is replaced stream gradually, be full of stream fully until detect liquid, liquid flows into waste liquid bottle 109 in this process, thereby finishes the importing that detects liquid.
(6) swivel tee valve 202 cuts out to detecting liquid liquid storage bottle 201, and stream is unimpeded between liquid driving pump 103 and detection liquid liquid storage bottle 201 downstreams; Swivel tee valve 108 to waste liquid bottle 109 is closed, stream is unimpeded between liquid driving pump 103, tissue culture chamber 105 and the test chamber 107, thereby form the loop of a sealing, detect liquid and driven by liquid driving pump 103, in tissue culture chamber 105 and test chamber 107, circulate; Detect the circulation of liquid in detecting the loop thereby finish.
(7) detect reaction signal by detector,, be delivered to computer 204, thereby finish the collection of initialize signal as initialize signal such as the variation of the signals such as absorbancy, fluorescence intensity, chemiluminescence intensity.
(8) detect liquid and driven by liquid driving pump 103, in tissue culture chamber 105 and test chamber 107, circulate the reaction times of regulation, the cell on detection agent and the anatomic implants is fully reacted; Detector detection reaction signal is delivered to computer 204 to signal, obtains the data of amount of viable cell through Computer Processing.
It should be noted that circulating of described detection liquid play the effect of mass transfer and mixing, make that to detect the liquid reaction more abundant, improve the signal of reflection, prevent that nutrition from exhausting, make things convenient for metabolic waste to transport.The described reaction times is 0.5-4 hour; Described reaction signal comprises: absorbancy, fluorescence intensity, chemiluminescence intensity etc.; Described signal detection comprises during reaction continuous detecting by the signal intensity of test chamber, perhaps the signal of test chamber during detection reaction terminal point.
(9) detect end, swivel tee valve 102 is unimpeded to stream between washing fluid liquid storage bottle 101 and the liquid driving pump 103; Swivel tee valve 202 cuts out to detecting liquid liquid storage bottle 201, and stream is unimpeded between washing fluid liquid storage bottle 201 and the liquid driving pump 103; Swivel tee valve 104 is unimpeded to stream between liquid driving pump 103, the tissue culture chamber 105, and stream is closed between 112 upstreams, tissue culture loop and the tissue culture chamber 105; Swivel tee valve 106 is unimpeded to stream between tissue culture chamber 105 and the test chamber 107, and stream is closed between 112 downstreams, 105 tissue culture loops, tissue culture chamber; Swivel tee valve 108 is unimpeded to stream between test chamber 107 and the waste liquid bottle 109, and stream is closed between waste liquid bottle 109 and the washing fluid liquid storage bottle 201; Detect the loop to the switching of flush loop thereby finish.
(10) open liquid driving pump 103, make washing fluid replace gradually other liquid of stream, until washing fluid is full of stream fully, liquid flows into waste liquid bottle 109 in this process, thereby finishes the flushing of tissue culture.
(11) the closing liquid driving pump 103, and rotation swivel tee valve 104 and 106 is unimpeded to stream between 112 upstream and downstream, tissue culture loop and the tissue culture chamber 105, and flushing and detection loop are closed, thereby reactor is switched to the tissue culture loop.
Embodiment 1 detects the amount of viable cell in the vascular tissue of cultivating in tissue culture procedures
(1) the swivel tee valve switches stream, and the tissue culture loop of tissue culture chamber 105 upstream and downstream is closed, and detection agent liquid storage bottle 201 is closed, and flush loop is unimpeded;
(2) open constant flow pump 103, washing fluid is flowed out from washing fluid liquid storage bottle 101, and the tissue culture of flowing through chamber 105, test chamber 107 flow into waste liquid bottle again, thereby finish the displacement to solution in tissue culture chamber and the culture, get rid of the interference of material in the nutrient solution (such as cast-off cells etc.) to detecting;
(3) after flushing is finished, the swivel tee valve switches stream, washing fluid liquid storage bottle 101 is closed, detect liquid liquid storage bottle 201 unobstructed, open constant flow pump 103, make and detect liquid from detecting the outflow of liquid liquid storage bottle, the tissue culture of flowing through chamber 105, test chamber 107 flow into waste liquid bottle again, detect liquid to the displacement of washing fluid in tissue culture chamber and the culture thereby finish, making liquid in the whole detection loop is the concentration homogeneous, the detection liquid of not diluted;
(4) the swivel tee valve switches stream, waste liquid liquid storage bottle 109 is closed, thereby detection liquid is circulated in loop, keep detecting and contain the composition (becoming to grade such as substratum, Hanks liquid) of keeping the cell eubolism in the liquid, the assurance cell is kept normal physiological status between detection period, guarantee the basic nutritional needs of cell between detection period.The loop closed circulation, the mass transfer of assurance reaction system prevents the topical substance overrich, affects reaction result.
(5) reaction signal that passes through detector of detection reaction initial time is delivered to computer 204 as initialize signal, thereby finishes the collection of initialize signal.
(6) allow detect the liquid specific time that circulates in the loop, when reaction finished, detector detection reaction signal was delivered to computer 204 to signal, obtained the data of amount of viable cell through Computer Processing.
(7) when reaction finishes, T-valve is switched to the washing lotion loop unimpeded;
(8) open constant flow pump 103, washing fluid is flowed out from washing fluid liquid storage bottle 101, the tissue culture of flowing through chamber 105 flows into waste liquid bottle 109 again, thereby finishes the flushing to tissue culture chamber and culture.
(9) close constant flow pump 103, the swivel tee valve cuts out and detects the loop upstream and downstream, switches to and cultivates the loop, makes the cultivation loop unimpeded; Continue tissue culture.
Embodiment 2 detects the cell viability on the microcarrier of suspension culture in tissue culture procedures
(1) the swivel tee valve switches stream, and the tissue culture loop of tissue culture chamber 105 upstream and downstream is closed, and detection agent liquid storage bottle 201 is closed, and flush loop is unimpeded;
(2) open constant flow pump 103, washing fluid is flowed out from washing fluid liquid storage bottle 101, cell retention device 501, the test chamber 107 of the tissue culture of flowing through chamber 105, the outlet of tissue culture chamber, flow into again waste liquid bottle, thereby finish the displacement to solution in tissue culture chamber and the culture, get rid of the interference of material to detecting in the nutrient solution;
(3) after flushing is finished, the swivel tee valve switches stream, washing fluid liquid storage bottle 101 is closed, detect liquid liquid storage bottle 201 unobstructed, open constant flow pump 103, make and detect liquid from detecting the outflow of liquid liquid storage bottle, the tissue culture of flowing through chamber 105, tissue culture chamber outlet cell retention device 501, test chamber 107 flow into waste liquid bottle again, detect liquid to the displacement of washing fluid in tissue culture chamber and the culture thereby finish, making liquid in the whole detection loop is the concentration homogeneous, the detection liquid of not diluted;
(4) the swivel tee valve switches stream, waste liquid liquid storage bottle 109 is closed, thereby detection liquid is circulated in loop, keep detecting and contain the composition (becoming to grade such as substratum, Hanks liquid) of keeping the cell eubolism in the liquid, the assurance cell is kept normal physiological status between detection period, guarantee the basic nutritional needs of cell between detection period.The loop closed circulation, the mass transfer of assurance reaction system prevents the topical substance overrich, affects reaction result.
(5) reaction signal that passes through detector of detection reaction initial time is delivered to computer 204 as initialize signal, thereby finishes the collection of initialize signal.
(6) allow detect the liquid specific time that circulates in the loop, during reaction, detector continuous detecting reaction signal is delivered to computer 204 to signal, obtains the data of cell viability through Computer Processing.
(7) when reaction finishes, T-valve is switched to flush loop unimpeded;
(8) open constant flow pump 103, washing fluid is flowed out from washing fluid liquid storage bottle 101, the tissue culture of flowing through chamber 105, flows into waste liquid bottle 109 again at the cell retention device 501 of tissue culture chamber outlet, thereby finishes the flushing to tissue culture chamber and culture.
(9) close constant flow pump 103, the swivel tee valve cuts out and detects the loop upstream and downstream, switches to and cultivates the loop, makes the cultivation loop unimpeded; Continue tissue culture.
Claims (9)
1. a viable cell proofing unit that is used for tissue engineering reactor comprises a detection loop in parallel with the cultivation loop of tissue engineering reactor and a flush loop in parallel with the cultivation loop of tissue engineering reactor.Detect loop and flush loop and combine by T-valve and pipeline, and detect the switching of loop and flush loop by valve opening and closing control.
Described flush loop comprises a washing fluid liquid storage bottle 101, a T-valve 102, a liquid driving pump 103,104, one tissue culture chambeies 105 of a T-valve, tissue culture 111, a T-valve 106, a test chamber 107,108, one waste liquid bottles 109 of a T-valve, pipeline 110 connects into a loop to above-mentioned parts;
Described detection loop comprises that is detected a liquid liquid storage bottle 201,103, one T-valve 104 of 202, one liquid driving pumps of a T-valve, a tissue culture chamber 105, tissue culture 111,106, one detectors 203 of a T-valve, the test chamber 107 that is connected with signal piping in the detector, a T-valve 108, a waste liquid bottle 109, pipeline 110 connects into a loop to above-mentioned parts, and detector is connected with a computer 204.
2. a kind of viable cell proofing unit for tissue engineering reactor according to claim 1 is characterized in that further comprising a flush loop in parallel with the cultivation loop of tissue engineering reactor:
Described tissue culture 111 is positioned at tissue culture chamber 105;
Described T-valve 104 and 106 lays respectively at the upstream and downstream in tissue culture chamber 105, finishes the switching of stream between tissue culture loop and detection or flush loop; T-valve 102 is positioned at washing fluid liquid storage bottle upstream, is used for the switching of control washing fluid liquid storage bottle;
Described liquid driving pump 103 is arranged between washing fluid liquid storage bottle 101 and the tissue culture chamber 105, be used for to drive liquid from washing fluid liquid storage bottle 101 flow through successively tissue culture chamber 105 and test chamber 107, flow into waste liquid bottle 109, finish the flushing to tissue culture.
3. a kind of viable cell proofing unit for tissue engineering reactor according to claim 1 is characterized in that further comprising a detection loop in parallel with the cultivation loop of tissue engineering reactor:
Described T-valve 202 is detecting between liquid liquid storage bottle 201 and the liquid driving pump 103, finishes stream and is detecting the loop, detecting the switching between circulation loop or the flush loop; T-valve 104 and 106 lays respectively at the upstream and downstream in tissue culture chamber 105, finishes the switching of stream between tissue culture loop and detection or flush loop; T-valve 108 is finished stream in the switching that detects between loop or the detection circulation loop between waste liquid bottle 109 and detection liquid liquid storage bottle 101;
Described detector is arranged on downstream, tissue culture chamber, for detection of signal intensity; Constant flow pump 103 driving detection liquid circulate in the loop of this sealing in testing process.
4. a kind of viable cell proofing unit for tissue engineering reactor according to claim 1, it is characterized in that further comprising and detect the loop and flush loop is combined by T-valve and pipeline, and detect the switching of loop and flush loop by valve opening and closing control:
A washing fluid liquid storage bottle 101, a T-valve 102, one is detected liquid liquid storage bottle 201, a T-valve 202, a liquid driving pump 103, a T-valve 104,106, one detectors 203 of 105, one T-valve in a tissue culture chamber, the test chamber 107 that is connected with signal piping in the detector, 108, one waste liquid bottles 109 of a T-valve, pipeline 110 connects into a loop to above-mentioned parts, detector is connected with a computer 204, by computer 204 control detectors and detect processing and the output of data;
Described detection liquid liquid storage bottle 201, washing fluid liquid storage bottle 101, waste liquid bottle 109 are set in turn in the upstream of liquid driving pump 103, link to each other with the pipeline of liquid driving pump 103 upstreams by T-valve 202,102,108 respectively, detect the switching of loop and flush loop by the open and close controlling of T-valve.
5. a kind of viable cell proofing unit for tissue engineering reactor according to claim 1 detects in the organizational project bio-reactor method of Growth of Cells and cytoactive on the anatomic implants, it is characterized in that comprising:
(1) closes tissue culture loop upstream and downstream
(2) switch to flush loop;
(3) flushing tissue culture;
(4) close flush loop, the open detection loop;
(5) detection reagent is injected in the tissue culture chamber;
(6) allow detection reagent circulate in detecting the loop, the optical density(OD) of detection original state or fluorescence intensity are as initial value (reference);
(7) allow detection reagent in detecting the loop, circulate, allow detection reagent react to specific time;
Optical density(OD) or fluorescence intensity when (8) detection reaction finishes with signal and the reference contrast that detects, are calculated amount of viable cell;
(9) switch to flush loop
(10) flushing tissue culture;
(11) close flush loop, switch to and cultivate the loop, continue tissue culture.
6. a kind of viable cell proofing unit for tissue engineering reactor according to claim 1, it is characterized in that the described pipeline 110 of described pipeline is comprised of flexible pipe or hard tube nontoxic, that can tolerate moist heat sterilization or chemosterilization such as oxyethane, alcohol sterilization, the diameter of pipe at 0.1mm between the 3mm.
7. a kind of viable cell proofing unit for tissue engineering reactor according to claim 1 is characterized in that described test chamber 107 is the cavitys that can carry out fluorescence, absorbancy or chemiluminescence detection; Detector 110 is the EM equipment module that can carry out fluorescence, absorbancy or chemiluminescence detection; Detector is connected with a computer 112, controls detectors by computer 112, and detects processing and the output of data.
8. a kind of viable cell proofing unit for tissue engineering reactor according to claim 1, it is characterized in that the reagent that described detection liquid can be or low toxicity nontoxic to cell comprises: the reagent such as the Alamar Blue that contain resazurin (Resazurin) dyestuff, the reagent that contains the diacetyl fluorescein(e) dye, the reagent that contains serum lactic dehydrogenase, the reagent that contain fluorine derivative (fluo derivative), furan derivatives (fura derivative), indole derivatives (indo derivate), rhod-2, calcium is green and quin-2 forms.And detect liquid and contain corresponding essential nutritive substance and the pH environment of histocyte maintenance metabolism, such as the cell culture fluid consistent with cell culture condition to be measured or D-Hanks liquid, PBS liquid etc., and these materials do not affect the carrying out of detection reaction and the detection of signal.
9. a kind of viable cell proofing unit for tissue engineering reactor according to claim 1, it is characterized in that described tissue culture 111 can be to be fixed in the tissue culture chamber of tissue engineering reactor, also can be that suspended state is cultivated in the tissue culture chamber.
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