JPWO2019217551A5

JPWO2019217551A5 -

Info

Publication number: JPWO2019217551A5
Application number: JP2020560492A
Authority: JP
Publication date: 2022-12-19
Anticipated expiration: 2039-05-08

Claims

An automated isolation device for isolating viable mitochondria from cells cultured in a living incubator, comprising:
a microprocessor configured to control operation of the automated isolation device;
one or more biological incubators (702, 860) configured to culture cells in preparation for mitochondrial isolation;
a pump (874) configured to transfer the cultured cells from the bioincubator to an incubation station (704, 814);
an incubation station (704, 814), comprising
a holder (212) for a solution containing viable mitochondria; and a cooling system (216) for cooling said holder, said cooling system being controlled by said microprocessor, in said incubation station. , an incubation station (704, 814) configured to incubate the cultured cells in a cold sterile solution in the presence of additives such as enzymes;
A first transfer system (706) comprising a first automated pump (230) configured to transfer a sterile solution containing the cells from the incubation station to a filtration station after incubation of the cells. a first transport system comprising:
a filtration station (708) comprising a series of filters (412, 414, 416), each successive filter having a smaller pore size than the preceding filter;
A second transfer system (712) comprising a second automatic pump (252), the second transfer system configured to transfer the filtered solution from the filtration station to the centrifugation station (714). , a second transfer system, wherein the filtered solution comprises purified mitochondria;
and a centrifugation station (714) for isolating purified mitochondria from the filtered solution.

2. The automated isolation device of claim 1, wherein said biological incubator consists of at least two biological incubators.

Each bioincubator comprises a roller flask (862) in which cells are cultured for growth, each roller flask is configured to be attached to an oxygen canister (864), and each roller flask is powered by an agitator (866). 3. The automated isolation device of claim 1 or 2, wherein the automated isolation device is controlled.

further comprising a spectrometer (710, 502) configured to respectively analyze a plurality of samples of cell cultures in said one or more biological incubators to determine the number of cultured cells per unit volume in each sample; The automated isolation device of any of claims 1-3, wherein the plurality of samples are removed from each of the one or more biological incubators at predetermined time intervals.

The pump (874) is configured to transport cultured cells in response to a demand for viable mitochondria, wherein operation of the pump (874) in response to demand is a unit in a given sample of the cell culture. 5. The automated isolation device of claim 4, determined based on the determined number of cultured cells per volume.

6. The automated isolation device of claim 4 or 5, wherein analysis of said sample of said cell culture and determination of the number of cultured cells per unit volume in said sample is a computer-controlled automated process.

the number of cultured cells per unit volume in a sample taken from a given one of said biological incubators (860) at each time interval determines the availability of a biological incubator to provide cultured cells; 7. The automatic separator according to any one of claims 4-6.

wherein said biological incubator (860) comprises at least two biological incubators, a given biological incubator comprising:
(i) is available for providing cultured cells when the number of cultured cells per unit volume in samples taken at consecutive time intervals has logarithmic growth;
(ii) not available if the number of cultured cells per unit volume is stable in samples taken at consecutive time intervals;
An automated isolation device according to claim 7.

An automated isolation device according to claim 7 or 8,
i) each biological incubator (860) includes a pneumatic valve (869) connecting the biological incubator to an incubation tube in an incubation station;
ii) an automated isolation device, wherein the available bioincubator pneumatic valve (869) is configured to be opened under computer control for a time sufficient to transfer a target number of cells to the incubation tube.

The automated isolation device of any of claims 1-9, wherein the sterile solution is solution A and the enzyme is subtilisin.

the filtration station includes a plurality of filters (412, 414, 416);
i) a first filter of said series of filters has a pore size between 30 μm and 50 μm;
ii) a second filter of said series of filters is positioned within the lumen of the filter unit adjacent to and below the first filter and has a pore size between 20 μm and 40 μm;
iii) a third filter of said series of filters is positioned within the lumen of the filter unit adjacent to and below the second filter and has a pore size between 10 μm and 30 μm;
iv) A fourth filter of said series of filters is positioned within the lumen of the filter unit adjacent to and below the third filter and has a pore size between 1 μm and 10 μm. 11. An automated isolation device according to any one of -10.

The automated isolation device of any of claims 1-11, comprising a spectroscopic measurement station (710, 502) configured to analyze the filtrate output from the filtration station.

the spectroscopic measurement station comprising:
a Raman spectrometer arranged to illuminate a cuvette (418) with a first wavelength of light, the cuvette being fluidly coupled to the output of a filtration station and controlled by the microprocessor; a Raman spectroscopy measurement station comprising a Raman spectrometer and a detector (504) located on the side of the cuvette opposite the Raman spectrometer and coupled to the microprocessor; 13. The automated isolation device of claim 12.

4. The automated isolation device of Claim 3, wherein the agitator is a hot plate agitator.

each biological incubator comprising a spectrometer and photomultiplier tube (PMT) (504) configured to analyze a sample of said cell culture;
i) the spectrometer is configured to emit light at wavelengths reflected by viable cultured cells contained in the sample of the cell culture;
ii) a cuvette (418) for containing said sample of said cell culture is fluidly coupled to the output of a bioincubator, positioned between said spectrometer and said PMT and transferred to an incubation station by a further transfer system; fluidly coupled to the inlet;
iii) when there are cultured cells in the sample, the PMT is configured to absorb less light and generate a smaller current than when there are no cultured cells in the sample;
An automated isolation device according to any of claims 1-14.

The automated isolation device of any of claims 1-15, wherein the pump configured to transport the cultured cells from the biological incubator to the incubation station is responsive to receiving a demand for viable mitochondria. .