Optical: systems and elements – Compound lens system – Microscope
Reexamination Certificate
2000-10-26
2003-03-25
Sikder, Mohammad (Department: 2872)
Optical: systems and elements
Compound lens system
Microscope
C359S392000, C359S393000, C359S394000, C359S395000
Reexamination Certificate
active
06538810
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to a single cell isolation apparatus in the form of a microscope slide, and more particularly to one for advantageous use in immunology applications, especially with respect to manipulation and handling of all biological cell types.
2. Description of Related Art
In the field of modern medicine, lab work and research often requires the use of a single cell. One such example of the type of work which requires a single cell is limiting dilution cloning (LDC). The purpose of LDC is to successfully isolate a single cell for each well in a microplate. While cloning by definition seeks to make a population of cells arise from a single cell, LDC can also be used to determine the frequency of a given event.
In LDC, a population of cells suspended in nutrient rich media is systematically diluted such that the concentration of cells per vial is reduced by factors of ten. For example, if an original cell population contains 1×10
6
cells per 1 ml of media, 10 &mgr;L of the solution is removed and placed into 1 ml of media. While theoretically the new concentration of cells should be 1×10
5
cells per ml, this is not always the case, and the problem can be exacerbated if the process of dilution is repeated several times. Statistically speaking, the chances of a “single hit” event, i.e. an event that requires only a single occurrence, like the growth of a single cell in a well, actually occurs 27% of the time based on experimental results although theoretically it should occur 100% of the time. In other words, if a dilution series is properly designed and carried out, 27% of the cultures will be positive, even though each well originally supposedly contained on average 1 cell per well.
Further complicating the situation is the fact that normally a single well may contain an average of 3-10 cells. This gives rise to the formation of a colony of cells. While an individual may be trying to isolate 1 specific type of cell that may exhibit a desirable trait, a well that contains 3-10 cells makes it virtually impossible to designate which cell is best for a particular application. A mathematical derivation and theoretical discussion associated with the above discussion is set forth in Mishell and Shiigi's “Selected Methods in Cellular Immunology.”
While microscope slides have existed for centuries, only fairly recently have they been used as a single cell isolation apparatus. Even so, the results up to this time point out the need for improvement. It is thus apparent that the need exists for a microscope slide which enables the effective isolation of a single cell. Moreover, based upon the size of the cells expected to be isolated using this invention, it would also be desirable for a single slide to permit a plurality of single cells to be isolated in distinct locations.
BRIEF SUMMARY OF THE INVENTION
There is disclosed a single cell isolation apparatus having a base plate with at least one micromachined aperture formed therein, a micromachined membrane having a tapered micromachined well sidewall, and a housing for holding the other components of the apparatus. The apparatus also may include a gel deposited above the base plate, a second membrane intermediate the base plate and the gel, and a filter above the gel, with the micromachined membrane being above the filter.
Each micromachined well associated with the apparatus has a tapered sidewall. The tapered sidewall has a upper diameter which corresponds to the average size of the type of cell attempting to be isolated through the use of the apparatus, and a lower diameter which is less than the upper diameter. In at least one embodiment of the invention, the tapered sidewall has a upper diameter of 15 &mgr;m. Generally, the tapered sidewall has a lower diameter of 1 &mgr;m, and in fact in the embodiment mentioned in the preceding sentence, the lower diameter of the tapered sidewall is 1 &mgr;m.
The gel component of the apparatus is a nitrocellulose gel. The filter is permeable to cellular products, and preferably is fabricated from paper. The second membrane provides fluid for the gel necessary for maintaining the gel's physical properties. The base plate preferably is a microscope slide.
There is also disclosed a microscope slide having at least one micromachined aperture drilled therein and with a transparent micromachined membrane having a tapered sidewall having an upper diameter and a lower diameter, with the upper diameter being greater than the lower diameter. In at least one embodiment, the tapered sidewall has a upper diameter of 15 &mgr;m. Further, in at least one embodiment, the tapered sidewall has a lower diameter it of 1 &mgr;m.
There is also disclosed a method for isolating a single cell comprising the steps of: (1) depositing a population of cells suspended in media onto the upper surface of a single cell isolation apparatus, with the single cell isolation apparatus having at least one micromachined well, wherein each micromachined well has a tapered sidewall, with each tapered sidewall having a upper diameter and a lower diameter with the lower diameter being less than the upper diameter, and (2) subjecting the single cell isolation apparatus to a vacuum, thereby holding a single cell in place in each well. Preferably, the upper diameter corresponds to the average size of the type of cell attempting to be isolated. The method also includes the additional step of washing away the free floating cells, with the free floating cells being those which are not being held in place in each well by the vacuum.
One objective of this invention is to provide a single cell isolation apparatus to permit the efficient isolation of a single cell.
Another objective of this invention is to provide a unique method for the holding of a single cell in place using a vacuum.
Another objective of this invention is to provide a single cell isolation apparatus which would permit a plurality of single cells to be isolated at the same time.
Still another objective of this invention is to provide an efficient method for isolation of single cells.
Other aspects and advantages of the instant invention will be appreciated from the following description, drawings, and the appended claims.
REFERENCES:
patent: 5231877 (1993-08-01), Henderson
patent: 5427975 (1995-06-01), Sparks et al.
patent: 6445109 (2002-09-01), Per.cedilla.in et al.
Kremblas, Foster Phillips & Pollick
Sikder Mohammad
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