CN103074213B - 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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- CN103074213B CN103074213B CN201210579150.3A CN201210579150A CN103074213B CN 103074213 B CN103074213 B CN 103074213B CN 201210579150 A CN201210579150 A CN 201210579150A CN 103074213 B CN103074213 B CN 103074213B
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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 the detection method that can detect living cell growth situation on tissue culture in organizational project bio-reactor or anatomic implants, the truth that in tissue culture procedures, cell grows and survives on some specific tissue engineering brackets or anatomic implants is detected for online or off-line continuous and quantitative, the influence factor of Growth of Cells and survival in assessment tissue culture, carries out quality control to sample.
Background technology
An important application of tissue engineering technique is exactly external structure anatomic implants, namely be by the seed cell of vitro culture with Method of Tissue Engineering external structure anatomic implants, be inoculated on the tissue engineering bracket of natural or synthetic, after cultivating in vitro, be transplanted to again in body, with the tissue of alternative disease damage.The treatment developing into congenital disorders and acquired disease of tissue engineering technique creates unprecedented chance.
Amount of viable cell on anatomic implants is a very important parameter, and it is analysis of cells growth, survival and the basis of metabolism, is the primary parameter controlling tissue culture procedures, is also the important indicator whether assessment anatomic implants has physiological function.In the culturing process of anatomic implants, often need the growing state understanding cell in time, quantitatively, to monitor culturing process.
But current 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 continuous print can not be carried out, detect dynamically.If want Continuous Observation Growth of Cells and survival condition, need to cultivate a large amount of sample simultaneously, in culturing process, take out culture 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, culturing process is complicated, cultivation is difficult, is subject to the restriction of cultivation scale, and continuous detecting and the assessment of Growth of Cells and survival in culturing process are just difficult to realization more.Therefore, field of tissue engineering technology in the urgent need to set up simple and quick, can at not damaging cells active and physiological function, carry out the quantitative proofing unit of viable cell and detection method under not interrupting the prerequisite of culturing process.
Chinese patent application (200680041273.4) discloses a kind of apparatus and method for detecting activity of living cells, uses 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 online or off-line, the device of cytoactive and growing state and corresponding detection method on the anatomic implants to cultivate in continuous print, quantitative detection tissue engineering reactor.Compared with prior art, the invention has the advantages that this device has a measure loop and a flush loop be in parallel with tissue culture loop be in parallel with tissue culture loop, cytoactive and cell growth status can be detected when not interrupting culturing process, and the harm of detection reagent to cell can be removed in time; Another advantage is the simple effectively easily enforcements of these apparatus and method, also not high to the requirement of tissue culturing equipment and test set.Its implementation result be to realize Growth of Cells in tissue culture procedures controlledization, can monitoring, be conducive to for anatomic implants clinical application, formulate relevant GLP and GMP specification foundation be provided.
Summary of the invention
According to an aspect of the present invention, provide a kind of can online or off-line, the device of cytoactive and growing state on the anatomic implants cultivated in continuous print, quantitative detection tissue engineering reactor, it is characterized in that comprising: a measure 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.Measure loop and flush loop are combined by valve and pipeline, and control the switching of measure loop and flush loop by valve opening and closing.
The described measure loop in parallel with the cultivation loop of tissue engineering reactor, is comprised one and detects liquid liquid storage bottle, be connected by pipeline with tissue culture chamber; A liquid driving pump is arranged on and detects between liquid liquid storage bottle and tissue culture chamber, flows in the loop for driving liquid; A detector, is arranged on downstream, tissue culture chamber, for change in detection signal; In testing process, constant flow pump drives detection liquid to circulate in the loop that this is closed.
The described flush loop in parallel with the cultivation loop of tissue engineering reactor, is comprised a washing fluid liquid storage bottle, is connected successively by pipeline with tissue culture chamber, detector; A waste liquid bottle being arranged on detector downstream, a constant flow pump is arranged between washing fluid liquid storage bottle and tissue culture chamber, for driving liquid to flow through tissue culture chamber and detector successively from washing fluid liquid storage bottle, flowing into waste liquid bottle, completing the flushing to measure loop;
An another aspect of the present invention, provide a kind of can be online, the method for cytoactive and growing state on the anatomic implants cultivated in continuous print, quantitative detection tissue engineering reactor, it is characterized in that comprising:
1, tissue culture loop upstream and downstream is closed
2, flush loop is switched to;
3, tissue culture is rinsed;
4, flush loop is closed, open detection loop;
5, detection reagent is injected in tissue culture chamber;
6, allow detection reagent circulate in measure loop, the optical density(OD) of detection original state or fluorescence intensity are as initial value (reference);
7, allow detection reagent circulate in measure loop, allow detection reagent react to specific time;
8, the optical density(OD) at the end of detection reaction or fluorescence intensity, by the signal that detects and control comparisons, calculate amount of viable cell;
9, flush loop is switched to
10, tissue culture is rinsed;
11, close flush loop, switch to and cultivate loop, continue tissue culture.
In one embodiment, described reaction reagent can be comprise the reagent of cytotoxic or low toxicity: the reagent containing resazurin (Resazurin) dyestuff is as Alamar Blue, reagent containing diacetyl fluorescein (FDA) dyestuff, reagent containing serum lactic dehydrogenase, the reagent of and quin-2 composition green containing fluorine derivative (fluo derivative), furan derivatives (fura derivative), indole derivatives (indo derivate), rhod-2, calcium.
Cell on tissue culture 111 described in an embodiment comprises: the various histocytes of original cuiture, the clone of cultured continuously, have the stem cell of multiple differentiation potential or single differentiation potential.These Growth of Cells are on tissue engineering bracket or on microcarrier.Described tissue culture 111 can be fixed in the tissue culture chamber of tissue engineering reactor, also can be that suspended state is cultivated in tissue culture chamber.
Accompanying drawing explanation
Fig. 1 schematically illustrates the structure of flush loop in viable cell proofing unit according to an embodiment of the invention;
Fig. 2 schematically illustrates the structure of measure loop in viable cell proofing unit according to an embodiment of the invention;
Fig. 3 schematically illustrates the structure of the viable cell proofing unit for tissue engineering reactor according to an embodiment of the invention, and wherein flush loop and measure loop are on a path;
Fig. 4 schematically illustrates the structure of T-valve according to an embodiment of the invention and the switching of path direction;
Fig. 5 shows the schema carrying out viable cell detection 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 a flush loop in parallel with tissue culture loop and 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, T-valve 106, a test chamber 107, a T-valve 108, a waste liquid bottle 109, pipeline 110 connects into a loop above-mentioned parts.For rinsing tissue culture 111 and measure loop before testing, remove the nutrient solution affecting detection reaction; Or wash tissue culture 111 and measure loop at the post-flush detecting amount of viable cell, remove and detect liquid, eliminate and detect 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 in tissue culture chamber 105 to cultivate, and also can be suspended in tissue culture chamber 105 to cultivate.
Described T-valve for controlling the opening and closing of stream or stream assembly, thus completes the switching of stream.Wherein said T-valve 104 and 106 lays respectively at the upstream and downstream in tissue culture chamber 105, completes 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, for controlling the opening and closing of washing fluid liquid storage bottle.
Described liquid driving pump 103 is arranged between washing fluid liquid storage bottle 101 and tissue culture chamber 105, tissue culture chamber 105 and test chamber 107 is flowed through successively from washing fluid liquid storage bottle 101 for driving liquid, flow into waste liquid bottle 109, complete the flushing to tissue culture.
In the specific implementation process of rinsing:
(1) between swivel tee valve 102 to washing fluid liquid storage bottle 101 and liquid driving pump 103, stream is unimpeded; Between swivel tee valve 104 to liquid driving pump 103, tissue culture chamber 105, stream is unimpeded, and between upstream, tissue culture loop 112 and tissue culture chamber 105, stream is closed; Between swivel tee valve 106 to tissue culture chamber 105 and test chamber 107, stream is unimpeded, and between downstream, tissue culture loop 112, tissue culture chamber 105, stream is closed; Between swivel tee valve 108 to test chamber 107 and waste liquid bottle 109, stream is unimpeded.
(2) open liquid driving pump 103, make washing fluid replace other liquid of stream gradually, until washing fluid is full of stream completely, liquid flows into waste liquid bottle 109 in the 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 a measure loop in parallel with tissue culture loop and 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 be connected with signal piping in detector, a T-valve 108, a waste liquid bottle 109, pipeline 110 connects into a loop above-mentioned parts, detector is connected with a computer 204, control detector by computer 204 and carry out detecting the process of data and output.T-valve 202, between detection liquid liquid storage bottle 201 and liquid driving pump 103, completes the switching of stream between measure loop, detection circulation loop or flush loop; T-valve 104 and 106 lays respectively at the upstream and downstream in tissue culture chamber 105, completes the switching of stream between tissue culture loop and detection or flush loop; T-valve 108 is at waste liquid bottle 109 and detect between liquid liquid storage bottle 101, completes the switching of stream between measure loop or detection circulation loop.
In the specific implementation process detected:
(1) swivel tee valve 202 is unimpeded to stream between detection liquid liquid storage bottle 201 and liquid driving pump 103, detect stream between liquid liquid storage bottle 201 and waste liquid bottle 109 to close, between swivel tee valve 104 to liquid driving pump 103, tissue culture chamber 105, stream is unimpeded, and between upstream, tissue culture loop 112 and tissue culture chamber 105, stream is closed; Between swivel tee valve 106 to tissue culture chamber 105 and test chamber 107, stream is unimpeded, and between downstream, tissue culture loop 112, tissue culture chamber 105, stream is closed; Between swivel tee valve 108 to test chamber 107 and waste liquid bottle 109, stream is unimpeded, and between waste liquid bottle 109 and detection liquid liquid storage bottle 201, stream is closed.
(2) open liquid driving pump 103, make detection liquid replace other liquid of stream gradually, until detect liquid to be full of stream completely, liquid flows into waste liquid bottle 109 in the process.
(3) swivel tee valve 202 cuts out to detection liquid liquid storage bottle 201, and between liquid driving pump 103 and detection liquid liquid storage bottle 201 downstream, stream is unimpeded; Swivel tee valve 108 to waste liquid bottle 109 is closed, between liquid driving pump 103 and test chamber 107, stream is unimpeded, thus the loop that formation one is closed, detect liquid to be driven by liquid driving pump 103, circulate in tissue culture chamber 105 and test chamber 107, the cell on the detection agent and anatomic implants of the reaction times internal volume of regulation is fully reacted; That detects liquid circulates the effect playing mass transfer and mixing, and make detection liquid react more abundant, improve the signal of reaction, prevent nutrition from exhausting, metabolic waste transports.
Wherein said pipeline 110 by nontoxic, moist heat sterilization can be tolerated or chemosterilization such as the flexible pipe of oxyethane, alcohol sterilizing or hard tube form, the diameter of pipe is between 0.1mm to 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 can carrying out fluorescence, absorbancy or chemiluminescence detection; Detector is connected with a computer 112, controls detector by computer 112, and carries out the process and the output that detect data.
Wherein said detection liquid can be comprise the reagent of cytotoxic or low toxicity: the reagent containing resazurin (Resazurin) dyestuff is as Alamar Blue, reagent containing diacetyl fluorescein(e) dye, reagent containing serum lactic dehydrogenase, the reagent of and quin-2 composition green containing fluorine derivative (fluo derivative), furan derivatives (fura derivative), indole derivatives (indo derivate), rhod-2, calcium.And detect liquid and contain the required nutritive substance of corresponding histocyte maintenance metabolism and pH environment, such as consistent with cell culture condition to be measured cell culture fluid 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 measure 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, 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 be connected with signal piping in detector, a T-valve 108, a waste liquid bottle 109, pipeline 110 connects into a loop above-mentioned parts, detector is connected with a computer 204, control detector by computer 204 and carry out detecting the process of data and output.
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, be connected respectively by the pipeline of T-valve 202,102,108 with liquid driving pump 103 upstream, by the open and close controlling measure loop of T-valve and the switching of flush loop.
Described flush loop and measure loop array mode can simplify viable cell proofing unit, in viable cell testing process, complete flushing and the detection of sample by stream easy switching.
As shown in Figure 4, T-valve according to an embodiment of the invention, by the rotation of T-valve inner spool, controls liquid flow path unimpeded on two or three directions, thus completes the switching of stream.
It should be noted that also can be used on loop and two two-way valves are set to replace described T-valve, as long as it can complete the process that corresponding stream switches.
As shown in Figure 5, tissue culture chamber 105 according to an embodiment of the invention, a cell retention device 501 is had in its exit, when anatomic implants suspension growth is in tissue engineering reactor, cell retention device 501 makes washing fluid, detect that the liquid such as liquid flows into detected downstream chamber 107 by tissue culture chamber 105 and cell is trapped within culture chamber.Described cell retention device can be core, filter or screen cloth, and it retains aperture at 1-10 μm.
As shown in Figure 6, the schema carrying out viable cell detection according to an embodiment of the invention.In the implementation process of the concrete detection of composition graphs 3 device:
(1) between swivel tee valve 104 to tissue culture upstream, loop 112 and tissue culture chamber 105, stream is closed, and between liquid driving pump 103, tissue culture chamber 105, stream is unimpeded; Between downstream, tissue culture loop 112, swivel tee valve 106 to tissue culture chamber 105, stream is closed, and between tissue culture chamber 105 and test chamber 107, stream is unimpeded; Thus close tissue culture loop.
(2) between swivel tee valve 102 to washing fluid liquid storage bottle 101 and liquid driving pump 103, stream is unimpeded; Swivel tee valve 202 cuts out to detection liquid liquid storage bottle 201, and between washing fluid liquid storage bottle 101 and liquid driving pump 103, stream is unimpeded; Between swivel tee valve 108 to test chamber 107 and waste liquid bottle 109, stream is unimpeded, and between waste liquid bottle 109 and washing fluid liquid storage bottle 201, stream is closed; Thus make flush loop unimpeded.
(3) open liquid driving pump 103, make washing fluid replace other liquid of stream gradually, until washing fluid is full of stream completely, liquid flows into waste liquid bottle 109 in the process, thus completes the flushing of tissue culture.
(4) swivel tee valve 202 is unimpeded to stream between detection liquid liquid storage bottle 201 and liquid driving pump 103, detects stream between liquid liquid storage bottle 201 and washing fluid liquid storage bottle 101 and closes; Between swivel tee valve 108 to test chamber 107 and waste liquid bottle 109, stream is unimpeded, and between waste liquid bottle 109 and washing fluid liquid storage bottle 101, stream is unimpeded; Swivel tee valve 102 cuts out washing fluid liquid storage bottle 101, and between washing fluid liquid storage bottle 101 and liquid driving pump 103, stream is unimpeded; Thus complete the switching of flush loop to measure loop.
(5) open liquid driving pump 103, make detection liquid replace other liquid of stream gradually, until detect liquid to be full of stream completely, liquid flows into waste liquid bottle 109 in the process, thus completes the importing detecting liquid.
(6) swivel tee valve 202 cuts out to detection liquid liquid storage bottle 201, and between liquid driving pump 103 and detection liquid liquid storage bottle 201 downstream, stream is unimpeded; Swivel tee valve 108 to waste liquid bottle 109 is closed, liquid driving pump 103, between tissue culture chamber 105 and test chamber 107, stream is unimpeded, thus the loop that formation one is closed, detect liquid and driven by liquid driving pump 103, circulate in tissue culture chamber 105 and test chamber 107; Thus complete the circulation of detection liquid in measure loop.
(7) detect the reaction signal by detector, if the change of the signals such as absorbancy, fluorescence intensity, chemiluminescence intensity is as initialize signal, is delivered to computer 204, thus completes the collection of initialize signal.
(8) detect liquid to be 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 anatomic implants is fully reacted; Detector detection reaction signal, signal transmission to computer 204, obtains the data of amount of viable cell through computer disposal.
What it should be noted that described detection liquid circulates the effect playing mass transfer and mixing, makes detection liquid react more abundant, improves the signal of reflection, prevent nutrition from exhausting, facilitate 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 comprise during reaction continuous detecting by the signal intensity of test chamber, or only detection reaction terminal time test chamber signal.
(9) detect end, between swivel tee valve 102 to washing fluid liquid storage bottle 101 and liquid driving pump 103, stream is unimpeded; Swivel tee valve 202 cuts out to detection liquid liquid storage bottle 201, and between washing fluid liquid storage bottle 201 and liquid driving pump 103, stream is unimpeded; Between swivel tee valve 104 to liquid driving pump 103, tissue culture chamber 105, stream is unimpeded, and between upstream, tissue culture loop 112 and tissue culture chamber 105, stream is closed; Between swivel tee valve 106 to tissue culture chamber 105 and test chamber 107, stream is unimpeded, and between downstream, tissue culture loop 112, tissue culture chamber 105, stream is closed; Between swivel tee valve 108 to test chamber 107 and waste liquid bottle 109, stream is unimpeded, and between waste liquid bottle 109 and washing fluid liquid storage bottle 201, stream is closed; Thus complete the switching of measure loop to flush loop.
(10) open liquid driving pump 103, make washing fluid replace other liquid of stream gradually, until washing fluid is full of stream completely, liquid flows into waste liquid bottle 109 in the process, thus completes the flushing of tissue culture.
(11) closing liquid driving pump 103, rotate swivel tee valve 104 and between upstream and downstream, 106 to tissue culture loop 112 and tissue culture chamber 105 stream unimpeded, rinse and measure loop closedown, thus reactor be switched to tissue culture loop.
Embodiment 1 detects the amount of viable cell in the vascular tissue of cultivation in tissue culture procedures
(1) swivel tee Vavle switching stream, 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) constant flow pump 103 is opened, washing fluid is flowed out from washing fluid liquid storage bottle 101, flow through tissue culture chamber 105, test chamber 107, flow into waste liquid bottle again, thus the displacement completed solution in tissue culture chamber and culture, in eliminating nutrient solution, material (as cast-off cells etc.) is to the interference detected;
(3) after having rinsed, swivel tee Vavle switching stream, washing fluid liquid storage bottle 101 is closed, detects liquid liquid storage bottle 201 unobstructed, open constant flow pump 103, detection liquid is flowed out from detection liquid liquid storage bottle, flow through tissue culture chamber 105, test chamber 107, then flow into waste liquid bottle, thus complete detection liquid to the displacement of washing fluid in tissue culture chamber and culture, the liquid in whole measure loop is made to be that concentration is homogeneous, the detection liquid of not diluted;
(4) swivel tee Vavle switching stream, waste liquid liquid storage bottle 109 is closed, thus detection liquid is circulated in loop, keep detecting in liquid containing the composition (as substratum, Hanks liquid composition etc.) maintaining cell eubolism, ensure that cell maintains normal physiological status between detection period, ensure the nutritional needs that cell is basic between detection period.Loop closed circulation, ensures the mass transfer of reaction system, prevents topical substance overrich, affect reaction result.
(5) reaction signal by detector of detection reaction initial time is as initialize signal, is delivered to computer 204, thus completes the collection of initialize signal.
(6) allow and detect liquid and to circulate in the loop specific time, at the end of reaction, detector detection reaction signal, signal transmission to computer 204, through the data of computer disposal acquisition amount of viable cell.
(7) at the end of reaction, T-valve is switched to washing lotion loop unimpeded;
(8) open constant flow pump 103, washing fluid is flowed out from washing fluid liquid storage bottle 101, flows through tissue culture chamber 105, then flow into waste liquid bottle 109, thus complete the flushing to tissue culture chamber and culture.
(9) close constant flow pump 103, swivel tee valve cuts out measure loop upstream and downstream, switches to and cultivates loop, make cultivation loop unimpeded; Continue tissue culture.
Embodiment 2 detects the cell viability on the microcarrier of suspension culture in tissue culture procedures
(1) swivel tee Vavle switching stream, 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) constant flow pump 103 is opened, washing fluid is flowed out from washing fluid liquid storage bottle 101, flow through cell retention device 501, the test chamber 107 of tissue culture chamber 105, the outlet of tissue culture chamber, flow into waste liquid bottle again, thus the displacement completed solution in tissue culture chamber and culture, in eliminating nutrient solution, material is to the interference detected;
(3) after having rinsed, swivel tee Vavle switching stream, washing fluid liquid storage bottle 101 is closed, detects liquid liquid storage bottle 201 unobstructed, open constant flow pump 103, detection liquid is flowed out from detection liquid liquid storage bottle, flow through tissue culture chamber 105, tissue culture chamber outlet cell retention device 501, test chamber 107, then flow into waste liquid bottle, thus complete detection liquid to the displacement of washing fluid in tissue culture chamber and culture, the liquid in whole measure loop is made to be that concentration is homogeneous, the detection liquid of not diluted;
(4) swivel tee Vavle switching stream, waste liquid liquid storage bottle 109 is closed, thus detection liquid is circulated in loop, keep detecting in liquid containing the composition (as substratum, Hanks liquid composition etc.) maintaining cell eubolism, ensure that cell maintains normal physiological status between detection period, ensure the nutritional needs that cell is basic between detection period.Loop closed circulation, ensures the mass transfer of reaction system, prevents topical substance overrich, affect reaction result.
(5) reaction signal by detector of detection reaction initial time is as initialize signal, is delivered to computer 204, thus completes the collection of initialize signal.
(6) allow and detect liquid and to circulate in the loop specific time, during reaction, detector continuous detecting reaction signal, signal transmission to computer 204, through the data of computer disposal acquisition cell viability.
(7) at the end of reaction, 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, flows through tissue culture chamber 105, the cell retention device 501 of tissue culture chamber outlet, flow into waste liquid bottle 109 again, thus complete the flushing to tissue culture chamber and culture.
(9) close constant flow pump 103, swivel tee valve cuts out measure loop upstream and downstream, switches to and cultivates loop, make cultivation loop unimpeded; Continue tissue culture.
Claims (11)
1., for a viable cell proofing unit for tissue engineering reactor, comprise a measure 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; Measure loop and flush loop are combined by T-valve and pipeline, and control the switching of measure loop and flush loop by valve opening and closing:
Described flush loop comprises a washing fluid liquid storage bottle 101, a T-valve 102, a liquid driving pump 103, a T-valve 104, tissue culture chamber 105, tissue culture 111, T-valve 106, a test chamber 107, a T-valve 108, waste liquid bottle 109, pipeline 110 connects into a loop above-mentioned parts;
Described measure loop comprises one and detects liquid liquid storage bottle 201, a T-valve 202, liquid driving pump 103, T-valve 104, a tissue culture chamber 105, tissue culture 111, T-valve 106, detector 203, the test chamber 107 be connected with signal piping in detector, a T-valve 108, a waste liquid bottle 109, pipeline 110 connects into a loop above-mentioned parts, and detector is connected with a computer 204;
In signal detection process, the T-valve 202 of described measure loop rotates to the closedown of liquid storage bottle detection liquid, between liquid driving pump 103 and detection liquid liquid storage bottle 201 downstream, stream is unimpeded, T-valve 108 rotates to waste liquid bottle 109 closes, liquid driving pump 103, between tissue culture chamber 105 and test chamber 107, stream is unimpeded, thus the loop that formation one is closed, detect liquid to circulate in loop, detection liquid is fully reacted, improve strength of signal, prevent nutrition from exhausting, promote that metabolic waste transports.
2. a kind of viable cell proofing unit for tissue engineering reactor according to claim 1, is characterized in that a described 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, completes 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, for controlling the opening and closing of washing fluid liquid storage bottle;
Described liquid driving pump 103 is arranged between washing fluid liquid storage bottle 101 and tissue culture chamber 105, tissue culture chamber 105 and test chamber 107 is flowed through successively from washing fluid liquid storage bottle 101 for driving liquid, flow into waste liquid bottle 109, complete 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 a described measure loop in parallel with the cultivation loop of tissue engineering reactor:
Described T-valve 202, between detection liquid liquid storage bottle 201 and liquid driving pump 103, completes the switching of stream between measure loop, detection circulation loop or flush loop; T-valve 104 and 106 lays respectively at the upstream and downstream in tissue culture chamber 105, completes the switching of stream between tissue culture loop and detection or flush loop; T-valve 108 is at waste liquid bottle 109 and detect between liquid liquid storage bottle 101, completes the switching of stream between measure loop or detection circulation loop;
Described detector, is arranged on downstream, tissue culture chamber, for change in detection signal; In testing process, liquid driving pump 103 drives detection liquid to circulate in the loop that this is closed.
4. a kind of viable cell proofing unit for tissue engineering reactor according to claim 1, it is characterized in that comprising measure loop further and flush loop is combined by T-valve and pipeline, and control the switching of measure loop and flush loop by valve opening and closing:
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 104,105, one, a tissue culture chamber T-valve 106, detector 203, the test chamber 107 be connected with signal piping in detector, a T-valve 108, waste liquid bottle 109, pipeline 110 connects into a loop above-mentioned parts, detector is connected with a computer 204, controls detector and carry out detecting the process of data and output by computer 204;
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, be connected respectively by the pipeline of T-valve 202,102,108 with liquid driving pump 103 upstream, by the open and close controlling measure loop of T-valve and the switching of flush loop.
5. a kind of viable cell proofing unit for tissue engineering reactor according to claim 1, it is characterized in that the viable cell proofing unit for tissue engineering reactor described in utilizing, detect the method for Growth of Cells and cytoactive on anatomic implants in organizational project bio-reactor:
(1) tissue culture loop upstream and downstream is closed
(2) flush loop is switched to;
(3) tissue culture is rinsed;
(4) flush loop is closed, open detection loop;
(5) detection reagent is injected in tissue culture chamber;
(6) allow detection reagent circulate in measure loop, the optical density(OD) of detection original state or fluorescence intensity are as initial value;
(7) allow detection reagent circulate in measure loop, allow detection reagent react to specific time;
(8) optical density(OD) at the end of detection reaction or fluorescence intensity, by the signal that detects and control comparisons, calculate amount of viable cell;
(9) flush loop is switched to
(10) tissue culture is rinsed;
(11) close flush loop, switch to and cultivate 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 described pipeline 110 is made up of flexible pipe that is nontoxic, that can tolerate moist heat sterilization or chemosterilization or hard tube, the diameter of pipe is between 0.1mm to 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 203 is the EM equipment module can carrying out fluorescence, absorbancy or chemiluminescence detection; Detector is connected with a computer 204, controls detector by computer 204, and carries out the process and the output that detect data.
8. a kind of viable cell proofing unit for tissue engineering reactor according to claim 1, it is characterized in that described detection liquid can be comprise the reagent of cytotoxic or low toxicity: the reagent containing resazurin (Resazurin) dyestuff, reagent containing diacetyl fluorescein(e) dye, reagent containing serum lactic dehydrogenase, the reagent of and quin-2 composition green containing fluorine derivative (fluo derivative), furan derivatives (fura derivative), indole derivatives (indo derivate), rhod-2, calcium; And detect liquid and contain the required nutritive substance of corresponding histocyte maintenance metabolism and pH environment, 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, is characterized in that described tissue culture 111 is fixed in the tissue culture chamber of tissue engineering reactor, or is suspended in tissue culture chamber.
10. a kind of viable cell proofing unit for tissue engineering reactor according to claim 1, it is characterized in that the described liquid that detects in testing process circulates in loop, detection liquid is fully reacted, and the time detecting liquid recycle stream dynamic is 0.5-4 hour.
11. a kind of viable cell proofing units for tissue engineering reactor according to claim 1, it is characterized in that described signal detection process to be included between the reaction solution reaction period monitoring continuously by the signal intensity of test chamber, also comprise the signal of test chamber during detection reaction terminal.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210579150.3A CN103074213B (en) | 2012-12-27 | 2012-12-27 | Living cell detection device and detection method for tissue engineering reactor |
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| CN109127554B (en) * | 2017-06-16 | 2023-09-22 | 中国人民解放军第五七一九工厂 | Cleaning device and cleaning method for oil laser particle counter sensor channel |
| CN108300659A (en) * | 2017-12-30 | 2018-07-20 | 宁波大学 | Cell incubator and its working method |
| CN110004056B (en) * | 2018-01-05 | 2020-12-11 | 北京航空航天大学 | Cell quantity and cell activity detection method and detection cavity device for tissue engineering reactor |
| CN117844629A (en) * | 2024-03-07 | 2024-04-09 | 柔脉医疗(深圳)有限公司 | Tissue culture liquid way detecting system |
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