This invention relates to a novel oocyte, embryo and specimen positioning apparatus and a method for inspection, observation, biopsy and the micromanipulation of oocytes, embryos and cells without the use of a second device, such as a holding pipette. The invention relates to the use of a series of structures within the apparatus that will allow the specimen to be placed into the apparatus and into the structures whereby the specimen is held in place may be easily located, and may be biopsied, manipulated or such without the use of a holding pipette. The apparatus allows the use of a single microtool, such as a micro injection pipette for the injection of materials, or a pipette to biopsy the specimen without a holding pipette to hold and stabilize the specimen during the procedure. The apparatus can be used for a wide range of specimens of animal and human cells, tissues, stem cells, embryos, oocytes and immature oocytes.
Historically, culture dishes have been used for many procedures in mammalian embryology and cell culture. One procedure may be for the growth and culturing of specimens in defined environments involving incubation. Another procedure involves observation, manipulation and handling the gametes and embryos. These procedures may be of varying lengths of time, some are short and involve a specific activity such as the addition or removal of cryoprotectant for freezing and thawing, or micromanipulation such as intra-cytoplasmic sperm injection (ICSI) for improving fertilization in men with infertility, or to biopsy the specimen for preimplantation genetic diagnosis (PGD) in couples at risk for passing a genetic anomaly on to their offspring. Most current dishes are flat and are not designed to accommodate any particular procedure.
Prior art micromanipulation patents such as in Wakayama et al, U.S. Pat. No. 6,641,526, describe methods of using a holding pipette for micromanipulation. Wakayama et al specifically describes a method for freeze-drying spermatozoa to obtain at least one reconstituted spermatozoon whose head (nucleus) is capable of fertilizing an oocyte to produce a live offspring, after microsurgical injection into an oocyte. In this method they use one holding pipette and one injection microneedle.
Games et al, U.S. Pat. No. 6,717,031, specifically uses a holding pipette for microinjection of DNA into the male pronucleus of a zygote to create transgenic mice. Lindenberg, U. S. patent application number 20050239040A again shows the use of a holding pipette for ICSI to improve in vitro fertilization. The procedures described in the aforesaid patents all utilize a holding pipette for the procedures.
Current prior art of Petrie dishes and embryological culture dishes do not show any dishes, which are designed to eliminate the use of a holding pipette. The prior art shows dishes which are designed to require the presence of a holding pipette.
Stevens, U.S. Pat. No. 5,484,731 describes a multi-well in vitrofertilization plate for general use using a holding pipette.
It would be highly desirable to have an apparatus that would allow one to perform procedures on embryos, cells, oocytes, and similar specimens without the use of a holding pipette to hold the specimen in place.
It would also be desirable to have an apparatus with a defined structure or configuration that would allow one to perform procedures on embryos, cells, oocytes and similar specimens without the use of a holding pipette to hold the specimen in place.
It would be highly desirable to have a Petrie dish or specific dish with a configuration or structure that serves as a microscopic specimen holding device. This would allow the user of the dish to manipulate the microscopic specimen, be it an embryo, and oocyte, a cell, or the like, with only one hand and only one instrument, and without the need of a holding pipette.
This invention relates to a culturing dish structure which enables a plurality of oocytes, cells and/or embryos to be held and positioned for injection or removal of a fluid without the need of a specimen holding pipette. The fluid can contain molecular reagents such as DNA, RNA, protein, cells and spermatozoa. The removed fluid can contain cells, fragments of cells, cytoplasm, nuclei and cellular organelles.
The individual oocytes, cells, or embryos (specimens) are kept separate from each other in open marked or unmarked well compartments that are located in the culturing dish. Each compartment comprises one or more finger like structures that can hold one oocyte, a clump of cells, or one embryo in a fixed position. The positioning of the specimens can be achieved using a single microneedle or microtool attached to a micromanipulation device. The configuration of the apparatus creates an area or lane having a flat bottom, and walls, whereby the lane has an opening and a narrowing between the walls.
The method and specimen-positioning apparatus of this invention may include a culturing container, such as a Petrie dish, in which the specimens may be captured for micromanipulation. The Petrie dish preferably contains a plurality of the specimen-holding finger-like structures which can be positioned in the Petrie dish in a predetermined pattern to form specimen capturing compartments in the Petrie dish. For example the capturing compartments can be positioned in the Petrie dish as a strip of compartments between the twelve o'clock and six o'clock positions, or in any other planned deployment in the dish. The compartment strip can be mirrored creating a duplicate row of finger-like projections in a mirror-like symmetry to be defined as mirrored structures which are composed of the same or varying components of the opposite side. The interdigitating narrowing areas allow for the positioning, holding, and micromanipulation of specimens, one in each compartment, without the need for a holding pipette to hold the specimens in place.
The method of this invention comprises the steps of placing the specimens in the container; moving each of the specimens into the lane structures of one of the interdigitating areas between the finger like projections; properly positioning each of the specimens with a microneedle connected to a micro manipulator; and using a suction device connected to this microneedle, when needed, for injection or biopsy. Conversely, the method of removing the specimens from the lane area will be comprised of the steps of: moving the microneedle adjacent to the specimen and pushing the specimen toward the narrowing end of the lane area; or using a standard pipette expelling culture fluid to direct the specimen toward the narrowing end of the lane. Cells or fragments removed can be placed in an alcove-like indentation in the structure for temporary storage to be used later or removed by a pipette. The reason for this alcove area is to place the biopsied cell or fragment away from the biopsied specimen so that both can be pipetted with a standard pipette separately allowing strict labeling and identification. The alcove is also the same focal length or the microscope in use, and in the same working plane as the micromanipulation device.
Additional variations of this invention can comprise marked capture compartments. These marked capture compartments can be identified by letters or numbers or symbols, for example, such as A, B, C, D, 1, 2, 3, 4, etc. inter alia. These markings can be etched or preprinted or simply written by the user.
The invention is structured in such a way that the dimensions of the area between the digits of the finger-like structures in the well can hold a mammalian oocyte, cell or embryo comfortably and securely so as to allow some compression and positioning of the specimen using a single micro manipulation micro pipette or micro needle tool. The compartments can be positioned in such a manner that oocytes and embryos will be safely separated allowing for stringent separation, manipulation, and administration. The individual oocytes, cell or embryos can be pipetted or otherwise moved into each compartment using any standard method and pipette tool which is normally used for embryological isolation and movement between wells and reagent micro droplets in a Petrie dish
The oocyte, cell or embryo can be removed from the restricting narrow areas of the compartment after the microprocedure by using a flow of fluid or culture medium released from a standard pipette or by gently probing the oocyte or embryo towards the tip of the narrowest end of the lane. A small alcove in each of the compartments may allow deposition and holding of biopsied cells or fragments from a biopsy micropipette after biopsy of the oocyte, cell or embryo. The biopsied cells or fragments can be pipetted safely using a narrow bored pipette. The dish may also contain separate wells for prepared samples of spermatozoa to be used for microinjection of one or more spermatozoa when the dish is used for intra-cytoplasmic sperm injection (ICSI). The dish may also contain other wells for washing oocytes or embryos before and after micromanipulation.
The apparatus of this invention may allow users to better and more effectively and efficiently perform the following procedures:
1) Intra-cytoplasmic sperm injection (ICSI) and other forms of assisted fertilization;
2) Polar body biopsy;
3) Assisted hatching, which is assisting the hatching process of a three day old embryo, or a blastocyst embryo, from the zona pellucida with a microtool, when the embryo is unable to hatch on its own;
4) Blastomere biopsy;
5) Blastocyst biopsy;
6) Manipulation of the zona pellucida (chemical, enzymatic or by laser);
7) Injecting solutions into the oocytes, blastomeres or blastocysts for transferring genes or other bio-molecules; and
8) Removing and injecting cytoplasm or organelles into oocytes or embryos.
These are some but not all procedures that the apparatus and method of this invention can be applied to.
These and other objects and advantages of the invention will become more readily apparent from the following detailed description of the invention, when taken in conjunction with the accompanying drawings in which:
Referring now to
Once the oocyte 24 is properly oriented, keeping the polar body 28 oriented away from the micro injection needle 26, the insemination of the sperm is performed as shown in
After insemination, the oocyte becomes an embryo 24′ as shown in
This dish structure allows each technician to use only one hand to perform a number of micromanipulation procedures on cells, embryos, and the like microscopic biological specimens, and does not require the use of a specimen holding straw with the other hand.
Including the zona pellucida, the average human oocyte is 150 microns in diameter, and falls with a range of 120-220 microns. The boundaries of the blastomeres in the embryo are well defined and their diameters range between 20 and 100 microns depending on their stage of cleavage. The early embryo has the same diameter as the oocyte because they are both contained within the zona pellucida.
Cells that can be biopsied are the polar bodies (by products of oocyte maturation) which range between 5 and 20 microns in diameter, and the blastomeres. Both oocytes and embryos may benefit from this procedure as the biopsied cell can be analyzed for genetic or metabolic content, for instance, chromosome number of genetic mutation as in preimplantation genetic diagnosis.
Techniques that can benefit from use with the dish structure of this invention include:
1) Intra-cytoplasmic sperm injection (ICSI) and other forms of assisted fertilization;
2) Polar body biopsy;
3) Assisted hatching, which is assisting the hatching process of a three day old embryo, or a blastocyst embryo, from the zona pellucida with a microtool when the embryo is unable to hatch on its own;
4) Blastomere biopsy;
5) Blastocyst biopsy;
6) Manipulation of the zona pellucida (chemical, enzymatic or by laser);
7) Injecting solutions into an oocyte, blastomere or blastocyst for transferring genes or other bio-molecules; and
8) Removing and injecting cytoplasm or organelles into oocytes or embryos.
While the invention has been described with respect to preferred embodiments, those skilled in the art will readily appreciate that various changes and/or modifications can be made to the invention without departing from the spirit or scope of the invention as defined by the appended claims.
This application claims the benefit of U.S. Ser. No. 12/592,630, filed Nov. 30, 2009; and U.S. Ser. No. 61/200,462, filed Dec. 1, 2008.
Number | Date | Country | |
---|---|---|---|
20150111297 A1 | Apr 2015 | US |
Number | Date | Country | |
---|---|---|---|
61200462 | Dec 2008 | US |
Number | Date | Country | |
---|---|---|---|
Parent | 12592630 | Nov 2009 | US |
Child | 13998307 | US |