Patent Application
20110256627
The present patent application claims the benefit of Taiwan Patent Application Number 98224454 filed Dec. 25, 2009.
The present invention relates to a cutting device for subculturing cells being applicable in next generation culturing of embryonic stem cells (such as human embryonic stem cells).
The human embryonic stem cells can differentiate into a variety of tissue cells in the human body, and is poised to play a key role in revolutionizing the current medical applications. The success of its related clinical applications is essentially hinged on the quantity and quality of cell culture used for laboratory tests, and thus improvements in culturing embryonic stem cells are crucial to clinical treatments, basic researches, and private pharmaceutical industries. Unlike most cells in culture, human embryonic stem cells are passaged as clumps. Single cell dissociation only decreases cell survival and doesn't contribute to successful subculture. Accordingly, known methods for cutting the cells include: (1) mechanical passage by glass pipet or rollers. It is time consuming, and the materials accompanying the use of rollers is highly expensive, and not environment-friendly. (2) enzyme passage by collagenase, trypsin or dispase. It is problematic in that the stability of chromosomes is potentially affected.
The inventor of the present invention has been working in the stem cell bank in Taiwan, and fully understands the negative effects the aforesaid shortcomings have on the research of the human embryonic stem cells; this invention was tested repeatedly and proposed in response to the shortcomings.
An object of the present invention is to provide a cutting device for subculturing cells being applicable in next generation culturing of embryonic stem cells (such as human embryonic stem cells), which does not have the shortcomings of the aforesaid methods.
Another object of the present invention is to provide a cutting device for subculturing cells that can be sterilized and re-used.
To achieve the above-mentioned objects of the invention, a cutting device for subculturing cells has been disclosed according to this invention, which includes a cutting brush, and the cutting brush comprises:
a handle comprising a holding portion for being held by human hands, and an inserting portion disposed at a front end of the holding portion;
a brush stem comprising a stem and a plurality of discrete upright wires disposed on the stem, and the wires may be disposed as one row or multiple parallel rows;
wherein the brush stem is mounted onto the inserting portion, and an angle between the brush stem and the holding portion ranges from 70 to 90 degrees.
Preferably, the cutting device of the invention further comprises a petri dish, and a length of the brush stem having a plurality of upright wires is smaller than a diameter of the petri dish.
Preferably, a distance between and two adjacent wires of the plurality of upright wires is smaller than 100 μm. More preferably, the distance between any two adjacent wires is greater than 20 μm.
Preferably, the angle between the holding portion and the inserting portion of the handle is adjustable. More preferably, an end of the inserting portion away from the brush stem is pivotally joined with a front end of the holding portion.
Preferably, the angle between the holding portion and the inserting portion of the handle is fixed and cannot be changed. More preferably, the holding portion and the inserting portion are formed as a unibody.
Preferably, the inserting portion has a mounting hole, and the brush stem is inserted and joined thereinto.
Preferably, the holding portion and the inserting portion are formed linearly, and an angle between an insertion direction of the mounting hole and the inserting portion ranges from 70 to 90 degrees.
Preferably, an angle between the holding portion and the inserting portion ranges from 70 to 90 degrees, and an insertion direction of the mounting hole is parallel to the inserting portion.
Preferably, each of the wires is of a diameter ranging from 0.1-10 μm, and a length ranging from 0.05-5 mm.
Preferably, the stem of the brush stem is Z-shaped, wherein the Z-shaped stem has a first horizontal portion inserted into and joined with the mounting hole, and a second horizontal portion having the plurality of discrete upright wires disposed thereon; a front end of the first horizontal portion is joined to a rear end of the second horizontal portion by a vertical or arc portion.
The present invention also discloses a method for cutting blocks of embryonic stem cells (such as human embryonic stem cells) for subculturing by using the cutting device of the invention, comprising the step of using the plurality of discrete upright wires to horizontally slice through a block of embryonic stem cells (such as human embryonic stem cells) in a petri dish intended for subculturing, thereby dividing the block of embryonic stem cells into a plurality of smaller blocks of embryonic stem cells.
The invention relates to a device for subculturing embryonic stem cells, and more particularly to a device for subculturing human embryonic stem cells. The device mainly comprises a cutting brush having a handle and a brush stem. The handle is preferably a metal bar, and can be sterilized under high temperature and high pressure for reuse. The handle may also be disposable, and is preferably made from reusable materials, such as by thermoplastic injection molding. An end of the handle has a mounting hole disposed thereon, which allows the brush stem to be inserted and secured thereinto. The brush stem can either be secured into the mounting hole by the friction there between, or directly combined with an end of the handle by the aforesaid thermoplastic injection molding. Preferably, the brush stem has a stem made of metal and wires made of gamma-ray resistant materials (such as nylon), or the stem and wires of the stem brush can all be made of gamma-ray sterilization resistant materials. According to a preferred embodiment of the invention, a brush stem of adequate length can be used accordingly to work with petri dishes of different diameters, so as to facilitate operations of experiments. The adequate ratios between petri dish diameters and adequate brush stem lengths are listed as follows:
The distances between the wires of the brush stem is smaller than the diameter of a block of (human) embryonic stem cells to be cut for next generation culture, so as to evenly cut the block into a plurality of smaller blocks of embryonic stem cells. For instance, a block of human embryonic stem cells is ready for subculture when reaching 500 μm in diameter on average, therefore the distances between the wires of the brush stem should be smaller than 100 μm.
A cutting device for subculturing cells according to a first preferred embodiment of this invention is shown in
The handle 10 is comprised of a holding portion 11 for being held by human hands, and an inserting portion 12 disposed at a front end of the holding portion. The inserting portion 12 further includes a mounting hole 13 (shown as dotted lines). The first horizontal portion 21 of the brush stem is inserted and secured into the mounting hole 13. An angle between the second horizontal portion 23 of the brush stem and the holding portion 11 is approximately 90 degrees.
The petri dish 30 has a cell culture medium 50 and a block of embryonic stem cells 40 cultured in the cell culture medium therein. When operating, an operator holds the holding portion 11 by hand, and horizontally slices the plurality of discrete upright wires 24 through the block of embryonic stem cells 40, and then rotates the petri dish by 90 degrees before slicing horizontally through the block again, such that the block is cut into a plurality of smaller blocks of embryonic stem cells.
A cutting brush for subculturing cells according to a second preferred embodiment of this invention is shown in
A cutting brush for subculturing cells according to a third preferred embodiment of this invention is shown in
A cutting device for subculturing cells according to a fourth preferred embodiment of this invention is shown in
To test the efficacy of this innovative cell cutter, we split embryonic stem (ES) cell cultures into two groups. For the control groups, the ES cells were undergone conventional passage treatment. ES cells were grown on MEF feeders and maintained in DMEM/F12 (supplemented with 20% knockout serum replacement, 2 mM L-glutamine, 1% non-essential amino acids, 4 ng/ml human bFGF (all from Invitrogen), and 0.1 mM 2-mercaptoethanol) (Sigma). Culture medium was changed daily and subculture was performed every 4-6 days by 1 mg/ml collagenase IV (Invitrogen). After PBS washes, the cells were dispersed by repeated pipeting and re-plated onto freshly thawed MEF. For the cutter group, ES cells were treated under the same condition as the control group except that the cutting device of the present invention was used to replace repeated pipeting. All cells were maintained in a 5% CO2-humidified atmosphere at 37° C.
To test the pluripotency of ES cells in culture, we performed EB formation assay. ES cells were detached and dispersed into small clumps. The cell clumps were then cultured in bacterial-graded Petri dish (Corning Inc) for up to 7 days in the presence of DMEM supplemented with 10% FBS (Invitrogen). All cells were maintained in a 5% CO2-humidified atmosphere at 37° C.
Total RNA was isolated using the mirVana miRNA isolation kit (Invitrogen) according to the manufacturer's instructions. RNA quality and quantity were evaluated on a NanoDrop spectrophotometer. One microliter of cDNA sample was PCR amplified with gene-specific primers (Forward sequence: AGC GAA CCA GTA TCG AGA AC, Reverse sequence: TTA CAG AAC CAC ACT CGG AC) by using optimized PCR cycles to obtain amplified reactions in a linear range. GAPDH was used for the internal control reaction on the same sample.
ES cells at post passage day five were removed from the incubator and then the medium was withdrawn before been fixed with 4% paraformaldehyde (Sigma) for 5 min at room temperature. Alkaline phosphatase staining was carried out with an alkaline phosphatase staining kit (Millipore, SCR004) according to the manufacturer's protocol. For the immunofluorescence assay, we grew ES cells on feeders in 24-well plate until ready. After removing media, we fixed ES cells with 4% paraformaldehyde for 15 min at room temperature. Cells were washed with 0.5% Triton x-100/PBS, at room temperature for 15 minutes and then blocked with 5% goat serum for 1 hour at room temperature. Primary antibodies against Oct-4 (Abcam) were diluted in PBS at 1:400 dilutions and then reacted with ES cells for 1 hour, at 37° C. Washed the cells with PBS, 5 minutes each, 3 times at room temperature (RT). Fluorescein anti-rabbit IgG were dilated with PBS at 1:5000 dilutions (Counter stain with DAPI/PBS), 1 hour at 37° C. before microscopic observation.
In order to identify whether human embryonic stem cells cultured through the conventional passage tool and the cutter passage tool of the present invention remain indifferent or not, they were observed by a phase contrast microscope. The results are shown in
While preferred embodiments of the invention are described above, it will be apparent to one skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 98224454 | Dec 2009 | TW | national |
