Method and apparatus for producing age-synchronized cells

Chemistry: molecular biology and microbiology – Apparatus – Bioreactor

Reexamination Certificate

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C435S299100, C073S863830, C073S864330, C073S864340

Reexamination Certificate

active

06767734

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method and apparatus that facilitates the collection of age-homogenous cell population and the maintenance of age synchrony in the collected cell population. More particularly, the invention is directed to a process and equipment for producing truly age-homogeneous cell population by obtaining an initial cell population in a very short period of time and maintaining the age homogeneity of this population during the subsequent aging process by constant removal of their offspring.
2. Description of Related Art
(1) The existing methods for cell synchronization.
Since early 1950s, various efforts have been made for obtaining “synchronized” cell population. For comprehensive reviews, see examples by E. Zeuthen (Synchrony in Cell Division and Growth, Interscience Publishers, New York, 1964), C. E. Helmstetter (Meth. Enzymol., 1, 327-363, 1969), and W. Krek and J. A. DeCaprio (Meth. Enzymol., 254, 114-124, 1995). In general, methods developed for cell synchronization can be classified as selection synchronization and induction synchronization.
In selection synchronization, a difference in the physicochemical properties of the cells in different divisional stages is often used as a basis for “cell cycle” (reproduction cycle) stage-specific separation. The physicochemical properties being utilized can be intrinsic cell properties such as the cell size and the cell density or artificially afforded properties such as the labeled molecules incorporated into the biomass or labeled molecules attached to the cell surface.
In induction synchronization, cells at different developmental stages are stopped at or induced into a predetermined developmental stage. Upon a desired time, these cells are allowed to proceed into subsequent development at the same release time to start the synchronization. The most frequently used agents for stopping or inducing cell development are those that interfere or promote “cell cycle” (cell reproduction cycle).
A special cell synchronization device called “baby machine” has been developed (C. E. Helmstetter and D. J. Cummings, Proc. Natl. Acad. Sci. USA, 50, 767-774, 1963; C. E. Helmstetter, New Biol., 3, 1089-1096, 1991; C. E. Helmstetter et al., J. Bacteriol., 174, 3445-3449, 1992). With this approach, baby cells released from the cells bound to a filter are collected and are allowed to grow together in further cultivation. It is hoped that the continuous cultivation of these baby cells should yield cell cycle synchrony for long time. But in reality, this expectation has never been realized, even when this method is used for synchronization of
Escherichia coli
and yeast (C. E. Helmstetter, New Biol., 3, 1089-1096, 1991; C. E. Helmstetter et al., J. Bacteriol., 174, 3445-3449, 1992).
A plate release technique has been used for synchronizing Caulobacter, an asymmetric bacterium that divides into a swarmer cell and a stalked cell (S. T. Degnen and A. Newton, J. Mol. Biol. 64, 671-680, 1972). Due to the adhesive property of the holdfast at the tip of the stalk, stalked cells attach to the surface such as the plate surface of the Petri dish and remain attached during their subsequent cell divisions. However, swarmer cells swim into the liquid phase once there are divided from the attached stalked cells because the motor activity of the polar flagellum on each swarmer cells. Thus, cell age synchronization of Caulobacter can be started simply by collecting swarmer cells released in a short period when they are divided from the adhered stalked cells. However, many studies have repeatedly shown that subsequent cultivation of these age-synchronous swarmer cells unavoidably leads to cell cycle asynchrony once the second cell cycle starts. This is because, while a stalked cell will divide soon after it finish the first cell cycle, a swarmer cell must grow into a stalked cell and then enters the next cell cycle. Thus, to achieve continuous cell cycle synchronization of Caulobacter, it is necessary to perform repeated density centrifugation to separate the two types of Caulobacter cells. This repeated centrifugation process is labor-intensive and time-consuming. For this reason, few studies on Caulobacter have extended into the second cell cycle of its life span.
Some methods have been developed for obtaining old cells of budding yeasts. One method is based on the size/density difference between the bigger mother cells and the smaller baby buds and requires successive repetition of rate-zonal sedimentation in sucrose density gradients to separate larger old cells from smaller young cells (N. K. Egilmez et al., J. Gerontol. Biol. Sci., 45, B9-B17, 1990). Another method depends on selectively labeling young cells with biotin and then obtains these biotin-labeled cells when they grow older through the binding between biotin and avidin, which is coated on magnetic beads (T. J. Smeal et al., Cell, 84, 633-642, 1996).
(2) The drawbacks of existing methods for cell synchronization.
It is well known that all existing methods of cell synchronization are inadequate for maintaining cell division synchrony for more than a few cell division cycles, whether the cells populations are prokaryotic unicellular microorganisms, eukaryotic unicellular microorganisms, or eukaryotic tissue cells of multicellular organisms (E. Zeuthen, Synchrony in Cell Division and Growth, lnterscience Publishers, New York, 1964; C. E. Helmstetter et al., J. Bacteriol., 174, 3445-3449, 1992). The underlying causes for such rapid deterioration of the synchrony in continuous culture of the initially synchronized cell population remains enigmatic.
A fundamental assumption made explicitly or inexplicitly for existing cell synchronization methods is that two cells formed from one cell are daughter cells of the same generation and of the same age (F. C. Neidhardt et al., Physiology of the Bacterial Cell: A Molecular Approach, Sinauer Associates, Inc., Sunderland, Mass., 1990; B. Alberts et al., Molecular Biology of the Cell, 3rd ed., Garland Publishing, Inc., New York, 1994). Because of this widely held but unproven assumption, it is generally believed that, once a cell population is obtained at or induced to the same cell division stage, it should automatically yield cells of the same division stage (often inappropriately called the cell age) during the subsequent cultivation.
However, this dogmatic view of cell life and cell synchronization is contradictory to the reality of many forms of cellular life and is also logically fallacious (S. V. Liu, Logical Biology, 2000, 5-16, 2000). A new model for cellular life proposes that the two cells formed from the division of one cell really belong to two successive generations and of different ages (S. V. Liu, Science in China, 42, 644-654, 1999). If the new model is correct, it means that the fundamental assumption made in most existing cell synchronization methods is invalid.
Collectively, the existing methods for cell synchronization often suffer one or more of the following drawbacks:
(a) The initial cell population used for starting cell synchronization often comprises cells of different ages. It has been widely believed that cells at the same divisional (reproduction) stages are of the same age. However, this assumption does not reflect all the reality and is logically invalid. For example, to say that all pre-divisional cells are of the same cell age is just like to say that all the pre-laboring mothers are of the same age. This statement is not true because even women of different ages can become pregnant at the same time and thus become “synchronous” in their reproduction stages.
(b) Cells belong to different generations are mixed in the continuous cultivation of the initially cell divisional stage- or cell age-synchronized cell population. The basic assumption of one mother cell divides into two daughter cells violates the fundamental principle of biological reproduction, which means a process for generation succession and genetic inheritance. Although final disproval of this

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