Chemical apparatus and process disinfecting – deodorizing – preser – Control element responsive to a sensed operating condition
Reexamination Certificate
2000-04-03
2001-10-30
Cooley, Charles E. (Department: 1723)
Chemical apparatus and process disinfecting, deodorizing, preser
Control element responsive to a sensed operating condition
C494S026000, C494S027000, C494S037000
Reexamination Certificate
active
06309606
ABSTRACT:
SUMMARY
The invention concerns a device and a method which allow the efficacious and rapid separation of organic cellular populations of different densities, from the dispersions which contain them, said device comprising an elongated chamber whose cross section decreases from the base towards the top of the device and said method comprising the introduction of a dispersion of a cell population into said chamber of the device previously filled with a liquid whose density varies from a maximum, near the device base, to a minimum near the top, subjecting the device with the cellular dispersion to centrifugation and collecting the separated fractions by introducing a very dense water immiscible liquid at the base of the separator device while simultaneously pumping a gas at the same pressure as said very dense liquid, to the device top so that the layered cellular fractions inside the chamber are expelled through at least one hole situated at an intermediate level of said chamber length.
PRIOR ART
The invention refers to both a device and method for the separation of human or animal cellular fractions of different densities, from cell populations present in cellular dispersions.
It is known that human or animal cells have differing densities (generally between 1.04 g/cm3 and 1.1 g/cm3) and that each cell type has its own characteristic density.
It is also known that it is often important and necessary to separate differing cell types for possible concentration or purification of the same, from a dispersion of a heterogeneous population of cells. This is fundamental in making the biochemical and functional analyses possible on both normal cells and neoplastic populations.
The finer and more careful the separation of different density cells, and the more rapid is the separation operation then the data obtained will be equally good and useful, with consequent benefits to the practical procedure.
If one considers, for example, that certain types of cells are present in the blood or human tissues in extremely reduced quantities, it is evident that from their effective and rapid identification, the possibility of chemical, biochemical or medical intervention, otherwise impossible or only hypothetical, could follow.
The most advanced cell separation technique in current use employs a separator device consisting of a casing with an internal cavity. The latter is laterally delimited by tapered surfaces, with the greatest cross section near the end or base from which a tube or duct end is connectable to a peristaltic pump. The cavity is tapered towards the opposite end or top of the casing, which has an outlet hole. Initially, a liquid is introduced into the base cavity of the separator device using the peristaltic pump and a gradient mixer. The liquid density slowly increases as the quantity of liquid in the cavity increases, until filling it. Thus, the separator device cavity is filled with a liquid which is practically made up of successive and continuous strata of liquid, increasing in density from the top to the base of the device cavity. At this point, the liquid supply is suspended and a dispersion of the cellular population to be separated into homogeneous cellular fractions by density is introduced through the hole in the top, until the upper cavity section is filled: at the same time as the dispersion introduction, a corresponding volume of the liquid first introduced is drawn off from the separator device base, so that the cellular population dispersion can fall into the device cavity.
The separator device is then closed in a centrifuge, with the rotation axis perpendicular to the separator device axis and near its top, during centrifugation. In the centrifuge, a very large force is transmitted to the liquid closed in the device (for example 400 g), resulting in the distribution of the cellular population dispersion in the liquid in the form of cellular bands (or strata) of differing densities.
The separator device is then removed from the centrifuge and a very dense water immiscible liquid is introduced into the base causing the expulsion (from the hole in the top of the same device) of the fractionated cellular dispersion as described: the desired number (for example 10 or 12) of distinct dispersion fractions with an increasing density gradient are extracted through the separator device top, the cells present in each fraction having different densities to the adjacent fractions.
The device and the methodology briefly outlined above, are described in detail by Giammaria Sitar and Piermaria Fomasari in HAEMATOLOGICA, Vol. 74, N1, January-February 1989; and by Pretlow II TG and Pretlow TP eds. Cell Separation: method and selected applications, New York, Academic Press, 1982 (5 volumes).
The recovery system of the different separated cell fractions described above, has its limits, however, and presents some serious problems. Infact, the collecting of the different and subsequent cell fractions is carried out through a small hole in the separator device top, under the push of a dense liquid which is pumped in at the device base. This causes a hydrodynamic disturbance in the different adjoining strata where the cell density is different, and causes the widening (well known to field technicians) of the strata or bands where the different density cells are collected or distributed, resulting in the contraction of the liquid strata separating the bands where are collected the cells of mainly homogeneous density and different from band to band, which can even lead to the remixing of the various cellular fractions that were separated during centrifugation. This phenomenon is more serious and unacceptable, the narrower the bands, in which the cells have been collected, and the smaller the distance separating one cell from another.
In order to try and minimise these problems, the pressurised liquid at the base of the separator device is introduced with extreme slowness, a fact which leads, however, to long and unacceptable times necessary for the collection of the different cellular fractions. See the publications of Giammaria Sitar and Piermaria Fomasari already mentioned; and TULP, Anal. Biochem. 1981, vol. 117, pages 354-365.
DESCRIPTION OF THE INVENTION
The main aim of the present invention is that of finding a device and method which allow the easy and very clear collection of cellular fractions which have been separated in the device after centrifugation.
Another aim is that of allowing the recovery of the different cellular fractions in shorter times than is currently possible, making the collection of useful data possible, which would otherwise have proved useless.
The above aims, along with others, are satisfied with a separator device with a base and top, containing an elongated chamber, whose cross section decreases from the base towards the device top, the latter containing at least a first channel, one end of which opens onto the inside of the said chamber near the said base, the other end connectable to a pressurised liquid source and a second channel, one end of which opens into the same chamber corresponding to the device top, characterised by the fact that in the said device there is at least an additional channel, one end of which opens along the said chamber length, the other end opening onto the exterior of the device.
The invention also concerns the separation method of cellular fractions from dispersions of human or animal cell populations, in which, first of all, the dispersion of a cell population is introduced into the separator device chamber, previously filled with a liquid whose density varies from a maximum near the device base and a minimum near the top. The device is then subjected to centrifugation so as to distribute the cells into distinct fractions with differing densities, the method being characterised by the fact that the separated fraction recovery occurs by introducing, in the device base, a very dense, water immiscible liquid while simultaneously pumping a gas to the top (at the same delivery pressure as the very dense liquid), so that the layered
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