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Researchers Submit Patent Application, "Cell Culture Apparatus, Apparatus for Long-Term Observation of Cell Culture, Method for Long-Term Cell...

June 17, 2014



Researchers Submit Patent Application, "Cell Culture Apparatus, Apparatus for Long-Term Observation of Cell Culture, Method for Long-Term Cell Culture, and Method for Long-Term Observation of Cell Cul

By a News Reporter-Staff News Editor at Life Science Weekly -- From Washington, D.C., NewsRx journalists report that a patent application by the inventors Wakamoto, Yuichi (Tokyo, JP); Hashimoto, Mikihiro (Tokyo, JP), filed on July 13, 2012, was made available online on June 5, 2014 (see also Patents).

No assignee for this patent application has been made.

News editors obtained the following quote from the background information supplied by the inventors: "Conventionally, it was difficult to continuously culture and observe cells placed in a container such as cell culture dish over many generations under a microscope because cells consume nutrition, waste products accumulate, and the environment around cells varies with the lapse of time. Furthermore, proliferation increases the number of cells in a container exponentially, which makes tracking and observing a certain cell difficult.

"Thus, the present inventors proposed a method for long-term culture of cells, for example, in which the cells are placed in a micrometer-size container and a part of the cells is removed out of the measurement system using a cell handling technique such as an optical tweezer (Non-patent document 1). However, in this method, since only a small number of cells can be transferred at one time and the experimenter has screen all the cells individually for removal, the work involves a great burden and the continuous culture has been practically limited to about ten generations at most.

"On the other hand, a method called 'mother machine' has recently attracted attentions as a method for long-term culture and observation of cells (Non-patent document 2). In this method, wide grooves (100 .mu.m width) and narrow whisker-like grooves (1 .mu.m width and 25 .mu.m length) are formed on a substrate. Cells are placed in the narrow grooves, and a culture solution is allowed to flow in the wide grooves to wash away and remove the unnecessary cells that are pushed out from the narrow grooves into the wide grooves as the cells proliferate. It is claimed that the cells in the narrow grooves can thus be cultured over many generations of 200 or more"

As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventors' summary information for this patent application: "Problems that the Invention is to Solve

"However, in the 'mother machine', one end of the narrow whisker-like groove is closed, and it is thus designed such that the cells remaining in the narrow grooves are always old cells (mother cells) when the unnecessary cells are continuously removed. That is, in the 'mother machine', there is no idea to eliminate the changes of physiological state associated with aging of the cultured cells remaining in the narrow whisker-like groove, and the 'mother machine' has a problem that the changes of cellular physiological state associated with aging are inevitable.

"Furthermore, in the 'mother machine', the culturing environment around cells in the narrow groove is controlled by exchange of the culture solution through diffusion from the wide groove. In this method, when the narrow groove is long, there is a problem that the environmental conditions considerably vary between the region closer to the wide groove and the region far from the wide groove. Accordingly, for example, when a response of cells to a drug is observed with this method, the response of the cultured cell could be different simply due to the difference in the environmental condition, and an accurate verification of cellular response could be difficult.

"The present invention was made in view of the above circumstances, and an object of the invention is to provide a cell culture apparatus, an apparatus for long-term observation of cell culture, a method for long-term cell culture and a method for long-term observation of cell culture, which are capable of continuously culturing and observing cells over long period of time under a uniform environmental condition without any variation in physiological state associated with aging of the cultured cells, and capable of tracing the history (genealogy) of a certain cell.

"Means for Solving the Problems

"The cell culture apparatus of the present invention is a cell culture apparatus comprising a cell culture substrate, a semipermeable membrane, and a culture solution supply means, for solving the above problems, wherein the cell culture substrate has, on the surface thereof, a narrow culture groove for holding and culturing cells and wide flow grooves for discarding the cells held and cultured in the culture groove, the both ends of the culture groove being connected to the flow grooves and the flow grooves being greater in width and depth than the culture groove; the semipermeable membrane is used so as to cover the culture groove and the flow grooves on the cell culture substrate; and the culture solution supply means is capable of continuously supplying culture solution to the cell culture substrate covered with the semipermeable membrane.

"In the cell culture apparatus, it is preferred that the semipermeable membrane is capable of covering the cell culture substrate via biotin-avidin binding.

"In the cell culture apparatus, it is more preferred that the culture solution supply means includes a liquid feeding pad.

"The apparatus for long-term observation of cell culture of the present invention is characterized by comprising the above-mentioned cell culture apparatus and a microscope observation means capable of observing cells on the cell culture substrate.

"In the apparatus for long-term observation of cell culture, it is preferred that an inverted microscope is used for microscopy.

"The method for long-term cell culture of the present invention is a method for culturing cells over a long period of time by the above-mentioned cell culture apparatus. In addition, the method is characterized by comprising the steps of: holding desired cells in the culture groove of the cell culture substrate; covering the culture groove and the flow grooves of the cell culture substrate with the semipermeable membrane; and continuously feeding culture solution to the cell culture substrate by the supply means to supply the culture solution to the cells held in the culture groove of the cell culture substrate through the semipermeable membrane, while discarding a part of cells in the culture groove to the flow grooves by the culture solution flowing in the flow grooves connected to the both ends of the culture groove.

"The method for long-term observation of cell culture of the present invention is a method for culturing and observing cells over a long period of time by the above-mentioned apparatus for long-term observation of cell culture. In addition, the method is characterized by comprising the steps of: holding desired cells in the culture groove of the cell culture substrate; covering the culture groove and the flow grooves of the cell culture substrate with the semipermeable membrane; continuously feeding culture solution to the cell culture substrate by the supply means to supply the culture solution to the cells held in the culture groove of the cell culture substrate through the semipermeable membrane, while discarding a part of cells in the culture groove to the flow grooves by the culture solution flowing in the flow grooves connected to the both ends of the culture groove; and observing the cells on the cell culture substrate by the microscope observation means.

"Advantage of the Invention

"According to the present invention, a long-term continuous culture becomes possible, and a growing state of cells can be observed over a long period of time while culturing the cells continuously under a uniform environmental condition or under an environmental condition with a regulated change, without involving any variation of the physiological state associated with aging of the cells to be cultured, and growth of a certain cell can be traced and observed over a long period of time. Accordingly, it becomes possible to measure a frequency distribution of cell size, a frequency distribution of growth rate, a frequency distribution of generation time, an autocorrelation function of expression level of a protein, and a cell genealogy.

BRIEF DESCRIPTION OF THE DRAWINGS

"FIG. 1 (A) is a partially enlarged view illustrating one embodiment of the cell culture substrate constituting the cell culture apparatus of the present invention, (B) is a cross-section of FIG. 1(A) taken along line A-A', and (C) is a cross-section of FIG. 1(A) taken along line B-B'.

"FIG. 2 It is a partially enlarged view illustrating another embodiment of the cell culture substrate constituting the cell culture apparatus of the present invention.

"FIG. 3 (A) is a general view illustrating one embodiment of the cell culture apparatus of the present invention with one part shown in cross-section, and (B) is a schematic diagram illustrating a method for using the semipermeable membrane.

"FIG. 4 They are pattern diagrams illustrating a state of cells when supplying culture solution to the cell culture substrate covered with the semipermeable membrane, in which (A) is a plane view, and (B) is a cross-sectional view.

"FIG. 5 It is a general view illustrating another embodiment of the cell culture apparatus of the present invention, with one part shown in cross section.

"FIG. 6 It is a general view illustrating one embodiment of the apparatus for long-term observation of cell culture of the present invention, with one part shown in cross section.

"FIG. 7 It is a part of continuous images recording a state of cells present in a culture groove of the cell culture substrate.

"FIG. 8 A graph by plotting the variation of cell size and the variation of the GFP expression level in the cell (intracellular concentration), over 55 generations in one cell line, obtained by the image analysis.

"FIG. 9 It is a graph showing a result of measurement of a frequency distribution of GFP expression level, which was estimated based on a measurement in which a time-lapse observation was made for a GFP-expressing E. coli, to determine the intracellular GFP average fluorescence brightness of all the cells at all the observation time point (Example 4).

"FIG. 10 It is a graph showing a result of a measurement of a frequency distribution of the cell size, by a time-lapse observation of the GFP-expressing E. coli (Example 5).

"FIG. 11 It is a graph showing a result of a measurement of a frequency distribution of the growth rate, by a time-lapse observation of the GFP-expressing E. coli (Example 6).

"FIG. 12 It is a graph showing a result of a measurement of a frequency distribution of the generation time, by a time-lapse observation of the GFP-expressing E. coli (Example 7).

"FIG. 13 It is a graph showing a result of a measurement of an autocorrelation function of the protein expression level, by a time-lapse observation of the GFP-expressing E. coli (Example 8).

"FIG. 14 It is a graph showing a result of a measurement of a frequency distribution of the cell division age, by a time-lapse observation of a GFP-expressing E. coli (Example 9).

"FIG. 15 It is a graph showing a result of a measurement of a cell genealogy observed in one observation groove, by a time-lapse observation of a GFP-expressing E. coli (Example 10).

"FIG. 16 (A) is a photograph showing a state of cells viewed from the upper side of a glass substrate, in the case of forming a culture groove having a groove depth smaller than the cell size, on the surface of the glass substrate, and (B) is a photograph showing a state of cells viewed from the upper side of a glass substrate, in the case of forming a culture groove having a groove depth equal to or more than twice as large as the cell size, on the surface of the glass substrate."

For additional information on this patent application, see: Wakamoto, Yuichi; Hashimoto, Mikihiro. Cell Culture Apparatus, Apparatus for Long-Term Observation of Cell Culture, Method for Long-Term Cell Culture, and Method for Long-Term Observation of Cell Culture. Filed July 13, 2012 and posted June 5, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=2275&p=46&f=G&l=50&d=PG01&S1=20140529.PD.&OS=PD/20140529&RS=PD/20140529

Keywords for this news article include: Patents, Escherichia, Cultured Cells, Enterobacteriaceae, Gammaproteobacteria.

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Source: Life Science Weekly


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