The method of quantitative determination of cell monolayer cultures and device for its implementation
The invention relates to immunology, in particular to assess the results of immunological tests. The proposed method and device for the quantitative determination of cell monolayer cultures. The culture is placed in a fixture to ensure the passage of the light flux through the studied culture and its directional reflection, then evaluate the optical density of the obtained reflection and its value determines the number of cells in the culture. The device is equipped with a locking device consisting of two parts: the bottom is a black screen with the slits and the top - cover with cylindrical projections, the ends of which are painted in matte white. The invention allows to count the number of cells without the use of specialized devices, to reduce error in the measurement. 2 C. p. F.-ly, 4 Il. , 1 table. The invention relates to immunology, in particular to assess the results of immunological tests.The purpose of the invention is the provision of opportunities for quantitative evaluation of cell colorimetric diagnostic systems based on colored monolayer cell cultures grown on planktonically device for the quantitative detection of the contents of the wells of flat-bottomed plates (patents US 4710031, US 5671290, US 4810096, JP 10274624 A2) and method colorimetric measurement of cell proliferation and cell viability (Mosman T. //J. immunol. Meth.-1983.-Vol.65.-P. 55-63; A. P. Shpakov, K. S. Pavlov, I. I. Bulychev // Clinical laboratory diagnostics - 2000. - 2.-S. 20-23; Matthews, N and Neale, M. L // Limphokines and Interferons, IRL Press, Washington, DC -1987.-P221).The closest analogue of the present invention described in the patent US 4710031 on "a Microtiter plate reader. invited reader for flat-bottomed plates, which allows visual examination of the contents of the array of holes of the die. The reader includes means to support the plate with a hole and has a light emitting surface located at the top. The area of light emission are arranged in such a way that correspond measured on the die holes. Input device signal light located underneath the die and thus to provide selective adjustment of the hole.However, all the proposed devices are not universal and are often closed or partially closed system. In addition, if uneven growth of a monolayer of cells on the surface of the hole, the use of such devices, which are used to measure a small part of the bottom area of the hole, can lead to significant errors th registration of the optical signal from the bottom of the flat-bottomed strips, used in cell colorimetric analysis with any size hole.The reading of optical information with flat-bottomed wells was performed using the flatbed, introducing optical information in the computer software filters and programs, measuring the optical density of the obtained image.We used a flatbed scanner with an optical resolution of 400600 600600 dpi. The choice of this range, the resolution was conditioned by two factors: lower optical resolution for accurate recording optical density resolution is insufficient, and when more significantly increase the system requirements for the computer and increases the time for image analysis because many times the size of the file with the scanned image. Scan images produced in different modes, choosing the optimum. For fixing strip on the scanner bed and minimize optical distortion of the received image, which leads to a distortion of the results, we used a special fixture. The locking device consists of two parts. The lower part is black naprosy the ICA scanner. The upper part is a lid with 8 cylindrical protrusions arranged perpendicular to the plane of the cover and corresponding in size and location to the holes of the strip. Their ends facing to the bottom of the hole, painted in matte white. This device allows you to minimize optical distortion of the image obtained, which can be used to signal non-specialized scanner.To measure the number of cells in the samples of culture with a known content of cells after fixation and staining is placed in a fixture so that the bottom holes of the strip enters the slot bottom and the tabs of the upper part is fixed in the cavity of the holes to their bottom. The locking device is placed on the flatbed scanner so that the light beam from the illuminator scanner through the slots of the bottom part fell on cells located at the bottom of the hole, was reflected from them and were registered by the optical sensor of the scanner. The signal obtained from the sensor is fed into a computer, where it forms an image of the bottom holes of the strip with the cells, and then processed using the program SigmaScan Pro 5, which converts the total optical density of the image of the bottom of kathiraveli samples with a known number of cells, using calibration curves. Putting on these curves, the values of optical densities of the studied crops, gain values of the number of cells in them.The invention is illustrated by drawings, where: Fig. 1 shows the lower part with the slots of the clamping devices for flat-bottomed strips, top view; Fig. 2 - both parts of the locking devices: the bottom part with the slots and the upper part with a cylindrical protrusions; a side view in section along the centerline of Fig.3 - both parts of the locking device, a front view in section along the centerline of Fig. 4 - the upper part of the locking device, bottom view.In Fig.1 shows one design variation, the clamping devices for flat-bottomed wells. The lower part of the device consists of black opaque screen 1, which is cut along the axis of movement of the optical sensor of the scanner lock for flat-bottomed strip with slots for the bottom holes of the strip 3. Screen 1 has a size equal to the size of the scanner bed, which prevents light from external sources on the mirror of the optical system of the scanner and, further, on the optical sensor.In Fig.2 shows both parts of the locking device. Top sastopami 5, the ends are directed to the bottom of the hole, painted in matte white. After placing the strip in the retainer 2 and covering the upper part of the device, the cylindrical protrusions are in the wells of the strip and, thus, prevents the influence of external light and internal light reflection light scanner from the walls of the holes, which minimizes optical distortion of the received image of the bottom holes of the strip.The example In the wells of a standard 8-hole flat-bottomed strip was placed serial cultivation of the cell line EJ bladder cancer man.Cells were cultured 24 h in medium RPMI-1640 with the addition of glutamine and 10% fetal cattle serum in an atmosphere of 6% CO2. After cultivation, the cells were fixed with methanol and stained 0,16% kristallwelten 1.6% ethanol for 10 minutes and Then the dye was washed with excess water. Painted the monolayer was placed in a spot photometer "Multiscan EX" and took into account the reaction at a wavelength of 620 nm. Then the strip was placed in a fixture and scanned the image strip with a resolution of 400600 600600 dpi. The resulting image was analyzed using the software is eating green software filter. As a parameter for evaluation used an average optical density. The data on the transmission transformed according to the formula A=1/D, where a is the optical density in relative units, and D is the average transmittance in relative units. Data obtained at a resolution of 400600 600600 dpi, practically coincided. After comparing the obtained graphs revealed that both graphs (and from a scanner, and with "Multiscan EX") is uniformly monotone and allow you to use them as a calibration curve in immunological experiments. The difference consisted in the fact that the curve obtained with the "Multiscan EX, had a more logarithmic in nature, it is less convenient for measurements related to extreme regions of this curve.Three wells of another strip in the same way were placed in three different sample of cells: 150 000, 70 000, 20 000 ml Optical density of these holes, processed as well as holes of the curve, was measured using a scanner. The obtained optical density was plotted on the ordinate of the graph, which have been previously applied values of optical density of the points of the calibration curve. The obtained point samples were projected on the calibration curve and t is dealt concentrations of cells in the wells of the calibration curve. Thus, we obtained the point of intersection of these perpendiculars to the x-axis, the numerical value which is approximated by the cell concentration in the investigated points. The table presents data comparing the value of the number of cells in the test wells obtained by direct counting in the Goryayev camera and processing the scanned image.The table shows that the measurement error in the number of cells in the samples obtained by analyzing the scanned image does not exceed 7%. When measuring samples with the standard method is by direct counting in the camera Goryaeva Pets error of up to 10%. Thus, the proposed method gives results comparable with the standard, but in contrast, automated and allows to quantify cell monolayer cultures.
Claims1. The method of quantitative determination of cell monolayer cultures, characterized in that the culture grown in the wells of flat-bottomed optically transparent strip is placed in a fixture to ensure the passage of the light flux through the studied culture and its directed ">600 600600 dpi and its value determines the number of cells in the culture.2. Device for the quantitative determination of cell monolayer cultures containing flatbed scanner including an optical sensor, characterized in that it is provided with a locking device consisting of two parts: the lower part, which is a black opaque screen with slots for fixing the flat-bottomed strip with holes containing the tested cells, and the upper part, which is a cap with a cylindrical protrusions arranged so that their ends facing to the bottom of the hole and painted in matte white color, and the size of the protrusions corresponds to the size of the holes of the strip.
FIELD: biotechnology, in particular isolation of mesenchymal stem cells from human tissues for treatment of various diseases.
SUBSTANCE: human mesenchymal stem cells are isolated from fat tissue, decidual placenta membrane, placenta caul, or placenta chorial stroma. Human tissue is crushed and treated with collagenase solution in DMEM (Dulbecco's Modified Eagle Medium) wherein fat tissue, decidual placenta membrane or placenta caul is treated with type I collagenase, and placenta chorial stroma is treated with type IV collagenase. Obtained suspension is purified from erythrocytes with lysing reagent followed by filtration through filters with pore size of 100 mum and 10 mum.
EFFECT: increased cell suspension homogeneity, target product yield, and cell viability.
2 cl, 4 tbl, 4 ex