Patent Application
20120315702
Cell culturing of specific cell lines or cells with specific attributes is an important aspect of many research and commercial production endeavors. Having a ready supply of uniform cells or cells with one or more desirable characteristics for research and product development is important to the advancement of work along these lines. Many types of cells, including human stem cells, can be seeded onto a surface of a growth substrate or other surface and proliferate into a colony of cells that forms a layer on the surface. These types of cells tend to thrive in an environment where they grow in close proximity to other cells of the same type. Once the cell culture has proliferated so as to produce a desired amount of cells, they may then be harvested. However, for cells such as human stem cells, as the cell colony proliferates and grows, the density of the cell population increases and the cells may begin to differentiate. In some cases, undifferentiated cells are desired for harvesting, so it may be important to remove the cells from the culture prior to differentiation or have the ability to segregate the differentiated cells from the undifferentiated cells prior to harvesting the cells.
Currently, the segregation of divided portions of cells from a layer of a cell colony growing on a substrate has been carried out by a painstaking process that requires a great deal of manual dexterity. In this process, a tool having a very fine tip point is made by hand by heating a portion of a glass pipette until the portion of the glass pipette is molten and malleable. The ends of the glass pipette are then pulled apart axially until the molten portion draws down to a fine thread about 20 to about 100 microns in diameter. The fine thread portion is then broken off at a position having a desired diameter and the original diameter portion of the pipette forms a handle portion. A user can then use the fine tip portion of the pipette tool as a tool to cut squares into or otherwise segment the layer of cell culture on the growth substrate by forming a criss-cross pattern by hand or other suitable method. Once the layer of cell culture has been cut into divided portions, the user may select divided portions of cells having desired attributes for selective removal of the divided portion from the layer of cell culture and relocation to a new growth substrate or other destination. The size of the cell culture layer may be several square inches and be divided into as many as several hundred divided portions or more. As such, this is a tedious and time consuming process that must be carried out under a microscope by a skilled operator.
What has been needed are devices and methods to allow a user to separate or partition a cell culture layer into divided portions having cells with one or more desirable attributes or a desired state without the need for handmade tools and tedious time intensive processes. What has also been needed are devices and methods for processing cell culture layers, generally, in an efficient and reliable manner.
Some embodiments of a cell culture processing device include a roller body having a substantially cylindrical outer surface with a layer penetrating structure 16 disposed thereon. The layer penetrating structure may be configured to penetrate a cell culture layer and partition the layer into divided portions. A support structure may be coupled to the roller body and configured to allow rotation of the roller body about an axis that is substantially concentric with the cylindrical outer surface. For some embodiments, the layer penetrating structure includes a plurality of adjacent circumferential ridges which may be regularly spaced in an axial direction. For some embodiments, the layer penetrating structure includes ridges surrounding substantially closed boundaries disposed at regularly space intervals on the cylindrical outer surface. For some embodiments, the outer surface of the roller body includes an elastomeric material.
Some embodiments of a cell culture processing tool include a roller body having a substantially cylindrical non-adherent outer surface with a layer penetrating structure configured to penetrate and partition a cell culture layer into divided portions disposed thereon. An axle may extend coaxially through the roller body and may be configured to support smooth rotational movement of the roller body about a longitudinal axis of the roller body. A handle may be coupled to the axle so as to allow rotational movement of the roller body about a longitudinal axis of the roller body relative to the handle. For some embodiments, the layer penetrating structure includes a plurality of adjacent circumferential ridges which may be regularly spaced in an axial direction. For some embodiments, the layer penetrating structure includes ridges surrounding substantially closed boundaries disposed at regularly spaced intervals on the cylindrical outer surface. For some embodiments, the handle includes an elongate handle body with a deflected distal section that forms an angle with a nominal longitudinal axis of the elongate handle body with the axle being coupled to the deflected distal section.
Some embodiments of a robotic cell culture processing tool may include a three axis robotic positioning actuator and a controller coupled to the three axis robotic positioning actuator. The processing tool may also include a roller body having a substantially cylindrical non-adherent outer surface with a layer penetrating structure configured to penetrate and partition a cell culture layer into divided portions. A support structure may be coupled to the roller body and configured to allow rotation of the roller body about an axis that is substantially concentric with the cylindrical outer surface. The support structure may also be secured to a movable carrier of the three axis robotic positioning actuator. For some embodiments, the layer penetrating structure includes a plurality of adjacent circumferential ridges which are regularly spaced in an axial direction. For some embodiments, the layer penetrating structure includes ridges surrounding substantially closed boundaries disposed at regularly spaced intervals on the cylindrical outer surface. For some embodiments, the controller includes a processor which is programmed to controllably move the roller body in a pre-determined pattern of motion which may be at least one linear pass across a cell culture layer disposed in a cell culture dish.
Some embodiments of a method of processing a cell culture layer include separating the cell culture layer by advancing a roller body having a layer penetrating raised structure across the cell culture layer. The roller body may be advanced across the cell culture layer while applying a predetermined amount of force against the cell culture layer to cut through the cell culture layer and partition the cell culture layer into divided portions. For some embodiments, the layer penetrating structure includes ridges surrounding substantially closed boundaries disposed at regularly spaced intervals on the cylindrical outer surface of the roller body and a single pass of the roller body across the cell culture layer is used to separate cell culture layer into isolated divided portions. For some embodiments, the layer penetrating structure of the roller body includes circumferential ridges spaced axially from each other and at least two passes of roller body across the cell culture layer in at least two different directions are used to separate cell culture layer into isolated divided portions.
Some embodiments of a method of processing a cell culture include seeding a cell culture support substrate with a cells and allowing the cells of the cell line to proliferate on the cell culture substrate and form a cell culture layer disposed on the cell culture substrate surface. The cell culture layer may then partitioned by advancing a roller body having a layer penetrating raised structure across the cell culture layer while applying a predetermined amount of force against the cell culture layer to cut through the cell culture layer and partition the cell culture layer into divided portions.
Some embodiments of a method for passaging cells may include partitioning a cell culture layer with a cell culture processing tool by rolling a layer penetrating structure of the cell culture processing tool over the cell culture layer so as to partition the layer of cells into a plurality of isolated divided portions. An isolated divided portion of the layer of cells may be selected having cells with at least one predetermined characteristic. The isolated divided portion may then be transported to a new location the transported cells allowed to proliferate in the new location and generate a new cell culture layer of cells having the predetermined characteristic. For some embodiments, partitioning the cell culture layer may include partitioning the cell culture layer into isolated divided portions with a single pass of a roller body having a layer penetrating structure which comprises ridges surrounding substantially closed boundaries disposed at regularly space intervals on a cylindrical outer surface of the roller body. For some embodiments, the layer penetrating structure may include a plurality of adjacent circumferential ridges regularly spaced in an axial direction on a cylindrical outer surface of the roller body and partitioning the cell culture layer may include partitioning the cell culture layer into isolated divided portions with two passes of the roller body across the cell culture layer in different directions. Some embodiments may include repeating this passaging method for about 1 passage to about 1000 passages.
Some embodiments for a method of maintaining a cell line in a desired state may include partitioning a cell culture layer with a cell culture processing tool by rolling a layer penetrating structure of the cell culture processing tool over the cell culture layer so as to partition the layer of cells into a plurality of isolated divided portions. An isolated divided portion of the layer of cells may be selected having cells in a predetermined state. The isolated divided portion may then be transported to a new location and the transported cells allowed to proliferate in the new location and generate a new cell culture layer of cells having the predetermined state. For some embodiments, partitioning the cell culture layer may include partitioning the cell culture layer into isolated divided portions with a single pass of a roller body having a layer penetrating structure which comprises ridges surrounding substantially closed boundaries disposed at regularly space intervals on a cylindrical outer surface of the roller body. For some embodiments, the layer penetrating structure may include a plurality of adjacent circumferential ridges regularly spaced in an axial direction on a cylindrical outer surface of the roller body and partitioning the cell culture layer may include partitioning the cell culture layer into isolated divided portions with two passes of the roller body across the cell culture layer in different directions.
These features of embodiments will become more apparent from the following detailed description when taken in conjunction with the accompanying exemplary drawings.
Embodiments of the invention relate generally to the cultivation of cells. In particular embodiments, the invention relates to devices, compositions and methods for cell cultivation, including prolonged cell cultivation. In some embodiments, the invention relates to the maintenance of cells in a particular state over an extended period of time. For example, embodiments may be used to maintain cells in a state of non-differentiation or differentiation, over an extended period of time of about 1 day to about 5 years, more specifically, about 3 days to about 1 year, and even more specifically, about 5 days to about 1 month. For some embodiments, cells may be maintained in a desired state for about 1 passage to about 1000 passages, more specifically, about 5 passages to about 500 passages, and even more specifically, about 20 passages to about 100 passages.
Embodiments of cell culture processing tools discussed herein have the ability to partition a cell culture layer into divided portions, including isolated divided portions, each having substantially the same surface area without cross contaminating the cells of one divided portion with the cells of another divided portion. Cell culture processing tool embodiments discussed herein may cut or otherwise create a partition through a cell culture layer without picking up cells as a layer penetrating structure of a roller body of the tool advances across the cell culture layer. As such, it may be desirable for an outer surface of the roller body to have a substantially non-adherent surface that may also be sterile and inert to avoid contamination of the cells of the cell culture layer with contaminants that are either disposed on the roller body or emanate from the material of the roller body. Divided portions of the cell culture layer may need to be isolated from all adjacent portions of the cell culture layer and remain in contact with an optional support substrate after partitioning to allow the divided portions to be lifted or otherwise removed from the support substrate. As such, it may be desirable in some embodiments for the roller body and layer penetrating structure thereof to be configured to prevent skidding or sliding of the layer penetrating structure as it is advanced across a cell culture layer. Embodiments of the cell culture processing tool may be economical to manufacture and configured as single use devices.
The cell culture processing tool 10 includes a roller body 12 having a cylindrical non-adherent outer surface 14 with a layer penetrating structure 16 disposed on the outer surface 14 of the roller body 12. The layer penetrating structure 16 is configured to penetrate a cell culture layer and partition the cell culture layer into divided portions which may assume a variety of shapes and sizes. The roller body 12 in
Referring to
The handle 22 has a generally elongate cylindrical shape with a round transverse cross section and a waist portion 36 tapering to a reduced transverse diameter at about the middle point of the handle body between the proximal end of the handle and the distal ends of the legs 26 and 28 of the deflected distal section 24. The waist portion 36 provides a recess for an operator to grasp between thumb and forefinger to maintain control of the movement of the tool 10 in an axial direction while using the tool 10. Embodiments of the handle 22 may have a length of about 2 inches to about 10 inches, more specifically, about 3 inches to about 8 inches, and even more specifically, about 4 to about 7 inches, and a nominal transverse diameter or dimension of about 0.1 inches to about 0.7 inches, more specifically, about 0.2 inches to about 0.5 inches in the portion of the handle body proximal of the distal deflected section 24. The first and second legs 26 and 28 of the bifurcation in the deflected distal section 24 may have a length of about 0.2 inches to about 1.5 inches, more specifically, about 0.5 inches to about 1.0 inch. The length of the deflected distal section 24 may have a length of about 0.5 inch to about 2.5 inches, more specifically, about 1.0 inch to about 1.5 inches, and may form an angle of about 110 degrees to about 160 degrees, more specifically, about 120 degrees to about 150 degrees with respect to the nominal longitudinal axis 34 of the elongate handle body 22.
Referring to
The elastomer material of the roller body 12 is molded over the elongate cylindrical axle 20 in a monolithic structure which is secured in fixed concentric relation to the roller body 12. The elastomer provides a material that is temperature stable, moldable, inert and non-adherent. The elastomer may also be sterilized by methods such as gamma irradiation or other suitable methods. Suitable materials for the roller body 12, outer surface 14 of the roller body 12 and layer penetrating structure 16 of the roller body 12 may include elastomers such as silicone rubber, polyurethanes as well as other suitable materials that provide the above characteristics. The first end and second end of the axle 20 are sized to freely rotate within the recesses 30 at the distal ends of the respective legs 26 and 28 of the bifurcated structure of the handle 22. For such embodiments, the recesses 30 in the ends of the legs 26 and 28 of the bifurcated structure of the handle 22 may be cylindrical concentric holes that extend through the distal portions of the legs 26 and 28. The holes may be through holes or blind holes that open to an interior portion between the two legs 26 and 28 of the bifurcated structure. In order for the roller body 12 to rotate freely about the axle 20 during use in partitioning a cell culture layer, it has been found that for some embodiments, it is useful for the portions of the axle 20 that rotate within the recesses 30 to have a small outer diameter relative the outer diameter of the roller body 12.
This structure provides free rotational motion of the roller body 12 to prevent skidding, sliding or plowing of the layer penetrating structure 16 of the roller body 12 over a cell culture layer as the roller body 12 is being rotated and advanced across the cell culture layer. Sliding, skidding or plowing of the layer penetrating structure 16 across the cell culture layer may produce cross contamination or deformation of the cell culture layer as well as other undesirable effects. For such embodiments, the shear resistance or resistance to inelastic shear deformation of the cell culture layer in contact with roller body 12 should be greater than the frictional resistance to rotation of the roller body 12 at the outer surface 14 of the roller body 12. In addition, the frictional force in shear between the outer surface 14 of the roller body 12 and the cell culture layer must be greater than the resistance to rolling of the roller body 12 at the outer surface 14 thereof. For some embodiments, the axle 20 may have an outer diameter that is about 10 percent to about 25 percent of the outer diameter of the roller body 12, more specifically, about 12 percent to about 22 percent, and even more specifically, about 15 percent to about 20 percent. For some embodiments, the axle 20 may have an outer diameter of about 0.02 inch to about 0.08 inch, more specifically, about 0.03 inch to about 0.05 inch and may be made from a suitable inert high strength material such as stainless steel or the like.
For some embodiments, the material of the roller body 12 and of the layer penetrating structure 16 may have a shore hardness of about 60 A to about 80 A, more specifically, about 65 A to about 75 A, and even more specifically, about 68 A to about 72 A. For some embodiments, the circumferential ridges 38 may be about 0.003 inches high to about 0.015 inches high, more specifically, about 0.005 inches high to about 0.012 inches high, and even more specifically, about 0.007 inches high to about 0.010 inches high, as indicated by arrow 46 in
The partitioning outer edge 42 of the ridge 40, or individual axially adjacent circumferential ridges 38, may have a radius of curvature R as indicated in
Embodiments of the roller body 12, and layer penetrating structure 16 thereof, may have an axial length of about 0.2 inch to about 1 inch, more specifically, about 0.3 inch to about 0.5 inch, and even more specifically, about 0.35 inch to about 0.45 inch. The roller body 12 and layer penetrating structure thereof may have an outer diameter of about 0.05 inch to about 1 inch, more specifically, about 0.1 inch to about 0.5 inch, and even more specifically, about 0.15 inch to about 0.25 inch, for some embodiments. Embodiments of the roller body 12, and layer penetrating structure 16 thereof, may have an axial length of about 1 inch to about 3 inches, more specifically, about 1.5 inches to about 2.5 inches, and even more specifically, about 1.8 inches to about 2.2 inches. The roller body 12 and layer penetrating structure thereof may have an outer diameter of about 0.5 inch to about 2 inches, more specifically, about 0.6 inch to about 1.5 inch, and even more specifically, about 0.8 inch to about 1.2 inch, for some embodiments.
The ridges 38 of the layer penetrating structure 16, as shown in more detail in
For some embodiments, the threads cut into the interior surface of the mold may have a pitch in an axial orientation of about 50 threads per inch to about 150 threads per inch, more specifically, about 100 threads per inch to about 120 threads per inch, and even more specifically, about 105 threads per inch to about 110 threads per inch. The threads on the mold surface may also have a thread angle of about 40 degrees to about 80 degrees, more specifically, about 50 degrees to about 70 degrees, and even more specifically, about 55 degrees to about 65 degrees. The threads may have a thread height of about 0.005 inch to about 0.015 inch, more specifically, about 0.008 inch to about 0.012 inch.
Once the mold is formed, the elastomer of the roller body 12 may be injection molded into the mold over the axle 20 so as to economically form a high precision repeatable structure. The handle 22 may also be produced by injection molding of such materials as ABS plastic, polyurethane, or the like having a shore hardness of about 85 B to about 95 B, more specifically, about 86 B to about 94 B, and even more specifically, about 87 B to about 93 B, for some embodiments. Some flexibility of the handle during use may be beneficial for some embodiments. Such a structure may be produced inexpensively enough to allow the cell processing tool to be used as a single use or disposable device.
For the embodiment shown in
In particular, the ridge structures 58 surrounding the enclosed boundary portions 60 may be about 0.003 inches to about 0.015 inches high, more specifically, about 0.005 inches to about 0.012 inches high, and even more specifically, about 0.007 inches high to about 0.01 inches high, as shown by arrow 64 in
The roller body 54, and layer penetrating structure 56 thereof, may have an axial length of about 0.2 inch to about 1 inch, more specifically, about 0.3 inch to about 0.5 inch, and even more specifically, about 0.35 inch to about 0.45 inch for some embodiments. The roller body 54 and layer penetrating structure 56 thereof may have an outer diameter of about 0.05 inch to about 1 inch, more specifically, about 0.1 inch to about 0.5 inch, and even more specifically, about 0.15 inch to about 0.25 inch, for some embodiments. For some embodiments, the material of the layer penetrating structure 56, which may include an elastomer material such as silicone rubber, as well as other suitable materials, may have a shore hardness of about 60 A to about 80 A, more specifically, about 65 A to about 75 A, and even more specifically, about 68 A to about 72 A. In general, the roller body 54 may have features, materials and dimensions which are the same as or similar to those of roller body 12 discussed above.
In addition to the cell culture layer cells and support layer materials discussed above, other cells and support layer materials may also be useful. Examples of animal cell culture media that may be prepared and used with embodiments of the present invention include without limitation DMEM, RPMI-1640, MCDB 131, MCDB 153, MDEM, IMDM, MEM, M199, McCoy's 5A, Williams' Media E, Leibovitz's L-15 Medium, Grace's Insect Medium, IPL-41 Insect Medium, TC-100 Insect Medium, Schneider's Drosophila Medium, Wolf & Quimby's Amphibian Culture Medium, cell-specific serum-free media (SFM) such as those designed to support the culture of keratinocytes, endothelial cells, hepatocytes, melanocytes, etc., F10 Nutrient Mixture and F12 Nutrient Mixture. Other media, media supplements and media subgroups suitable for preparation by the invention are available commercially (e.g., from Invitrogen, Inc.; Rockville, Md., and Sigma; St. Louis, Mo.). Formulations for these media, media supplements and media subgroups, as well as many other commonly used animal cell culture media, media supplements and media subgroups are well-known in the art and may be found, for example in the GIBCO/BRL Catalogue and Reference Guide (Life Technologies, Inc.; Rockville, Md.) and in the Sigma Animal Cell Catalogue (Sigma; St. Louis, Mo.).
Cells may be cultured in undefined or defined media in certain embodiments. To overcome drawbacks of the use of serum or organ/gland extracts, a number of so-called “defined” media have been developed. These media, which often are specifically formulated to support the culture of a single cell type, contain no undefined supplements and instead incorporate defined quantities of purified growth factors, proteins, lipoproteins and other substances usually provided by the serum or extract supplement. Since the components (and concentrations thereof) in such culture media are precisely known, these media are generally referred to as “defined culture media.” Often used interchangeably with “defined culture media” is the term “serum-free media” or “SFM.” A number of SFM formulations are commercially available, such as those designed to support the culture of endothelial cells, keratinocytes, monocytes/macrophages, fibroblasts, chondrocytes or hepatocytes which are available from GIBCO/LTI (Gaithersburg, Md.). The distinction between SFM and defined media, however, is that SFM are media devoid of serum, but not necessarily of other undefined components such as organ/gland extracts. Indeed, several SFM that have been reported or that are available commercially contain such undefined components, including several formulations supporting in vitro culture of keratinocytes (Boyce, S. T., and Ham, R. G., J. Invest. Dermatol. 81:33 (1983); Wille, J. J., et al., J. Cell. Physiol. 121:31 (1984); Pittelkow, M. R., and Scott, R. E., Mayo Clin. Proc. 61:771 (1986); Pirisi, L., et al., J. Virol. 61:1061 (1987); Shipley, G. D., and Pittelkow, M. R., Arch. Dermatol. 123:1541 (1987); Shipley, G. D., et al., J. Cell. Physiol. 138:511-518 (1989); Daley, J. P., et al., FOCUS (GIBCO/LTI) 12:68 (1990); U.S. Pat. Nos. 4,673,649 and 4,940,666). SFM thus cannot be considered to be defined media in the true definition of the term.
Defined media can provide several advantages to the user. For example, the use of defined media facilitates the investigation of the effects of a specific growth factor or other medium component on cellular physiology, which may be masked when the cells are cultivated in serum- or extract-containing media. In addition, defined media typically contain much lower quantities of protein (indeed, defined media are often termed “low protein media”) than those containing serum or extracts, rendering purification of biological substances produced by cells cultured in defined media far simpler and more cost-effective. Some extremely simple defined media, which consist essentially of vitamins, amino acids, organic and inorganic salts and buffers have been used for cell culture. Such media (often called “basal media”), however, are usually seriously deficient in the nutritional content required by most animal cells. Accordingly, most defined media incorporate into the basal media additional components to make the media more nutritionally complex, but to maintain the serum-free and low protein content of the media. Examples of such components include serum albumin from bovine (BSA) or human (HSA); certain growth factors derived from natural (animal) or recombinant sources such as EGF or FGF; lipids such as fatty acids, sterols and phospholipids; lipid derivatives and complexes such as phosphoethanolamine, ethanolamine and lipoproteins; protein and steroid hormones such as insulin, hydrocortisone and progesterone; nucleotide precursors; and certain trace elements (reviewed by Waymouth, C., in: Cell Culture Methods for Molecular and Cell Biology, Vol. 1: Methods for Preparation of Media, Supplements, and Substrata for Serum-Free Animal Cell Culture, Barnes, D. W., et al., eds., New York: Alan R. Liss, Inc., pp. 23-68 (1984), and by Gospodarowicz, D., Id., at pp 69-86 (1984); see also US).
Any type of cell that can be cultured may be utilized in conjunction with embodiments of the invention described herein. Animal cells that can be used include, but are not limited to, cells obtained from mammals, birds (avian), insects or fish. Cell types or cells in desired states, such as differentiated states, that can be utilized include without limitation embryonic cells, stem cells, fetal cells and differentiated cells (e.g., from brain, eye, skin (e.g., dermal, sub-dermal, karatinocytes, melanocytes), trachea, bronchus, lung, heart, umbilical cord, cervix, ovary, testes fibroblast, blood). Cell types that can be utilized herein include fibroblast, epithelial and hematopoietic cells for example. Mammalian cells include without limitation rodent (e.g., mouse, rat, rabbit, hamster), canine, feline, monkey, ape and human cells. Mammalian cells that can be utilized include primary cells derived from a tissue sample, diploid cell strains, transformed cells or established cell lines (e.g., HeLa), each of which may optionally be diseased or genetically altered. Mammalian cells, such as hybridomas, CHO cells, COS cells, VERO cells, HeLa cells, 293 cells, PER-C6 cells, K562 cells, MOLT-4 cells, M1 cells, NS-1 cells, COS-7 cells, MDBK cells, MDCK cells, MRC-5 cells, WI-38 cells, SP2/0 cells, BHK cells (including BHK-21 cells) and derivatives thereof also may be used herein. Insect cells particularly suitable for use in forming such compositions include those derived from Spodoptera species (e.g., Sf9 or Sf21, derived from Spodoptera frugiperda) or Trichoplusa species (e.g., HIGH FIVE™ or MG1, derived from Trichoplusa ni). Cells from cell lines or from primary sources can be useful for certain embodiments. Tissues, organs, organ systems and organisms derived from animals or constructed in vitro or in vivo using methods routine in the art may similarly be used. Cells may be utilized herein in a variety of medical (including diagnostic and therapeutic), industrial, forensic and research applications requiring ready-to-use cultures of animal cells in serum-free media.
Animal cells for culturing and use in conjunction with embodiments of the present invention may be obtained commercially, for example from ATCC (Rockville, Md.), Cell Systems, Inc. (Kirkland, Wash.), Clonetics Corporation (San Diego, Calif.), BioWhittaker (Walkersville, Md.), or Cascade Biologicals (Portland, Oreg.). Alternatively, cells may be isolated directly from samples of animal tissue obtained via biopsy, autopsy, donation or other surgical or medical procedure. Names of cells available from such commercial sources are incorporated by reference herein.
Cells may be derived from tissue in certain embodiments. Tissue generally is handled using standard sterile technique and a laminar flow safety cabinet. In the use and processing of all human tissue, the recommendations of the U.S. Department of Health and Human Services/Centers for Disease Control and Prevention should be followed (Biosafety in Microbiological and Biomedical Laboratories, Richmond, J. Y. et al., Eds., U.S. Government Printing Office, Washington, D.C. 3rd Edition (1993)). The tissue often is cut into small pieces (e.g., 0.5.times.0.5 cm) using sterile surgical instruments. The small pieces generally are washed twice with sterile saline solution supplemented with antibiotics as above, and then may be optionally treated with an enzymatic solution (e.g., collagenase or trypsin solutions, each available commercially, for example, from GIBCO/LTI, Gaithersburg, Md.) to promote dissociation of cells from the tissue matrix.
The mixture of dissociated cells and matrix molecules are washed twice with a suitable physiological saline or tissue culture medium (e.g., Dulbecco's Phosphate Buffered Saline without calcium and magnesium). Between washes, the cells are centrifuged (e.g., at 200.times.g) and then resuspended in serum-free tissue culture medium. Aliquots are counted using an electronic cell counter (such as a Coulter Counter). Alternatively, the cells can be counted manually using a hemocytometer.
The isolated cells can be plated according to the experimental conditions determined by the investigator. Optimal plating and culture conditions for a given animal cell type can be determined by one of ordinary skill in the art using only routine experimentation. For routine culture conditions, using the present invention, cells can be plated onto the surface of culture vessels without attachment factors. Alternatively, the vessels can be pre-coated or contacted with natural, recombinant or synthetic attachment factors or peptide fragments (e.g., collagen or fibronectin, or natural or synthetic fragments thereof). Isolated cells can also be seeded into or onto a natural or synthetic three-dimensional support matrix such as a preformed collagen gel or a synthetic biopolymeric material. Use of attachment factors or a support matrix with the medium of the present invention will enhance cultivation of many attachment-dependent cells in the absence of serum supplementation. Cell seeding densities for each experimental condition may be optimized for the specific culture conditions being used. For routine culture in plastic culture vessels, an initial seeding density of 1-5.times.10.sup.6 cells per cm.sup.2 is preferable.
Mammalian cells typically are cultivated in a cell incubator at about 37.degree. C. The incubator atmosphere should be humidified and should contain about 3-10% carbon dioxide in air, although cultivation of certain cell lines may require as much as 20% carbon dioxide in air for optimal results. Culture medium pH often is in the range of about 7.1-7.6, preferably about 7.1-7.4, and most preferably about 7.1-7.3. Cells in closed or batch culture may undergo complete medium exchange (i.e., replacing spent media with fresh media) about every 1-2 days, or more or less frequently as required by the specific cell type. Cells in perfusion culture (e.g., in bioreactors or fermenters) will receive fresh media on a continuously recirculating basis.
In order to passage or otherwise cultivate a cell culture and produce a large supply of cells within a particular cell line or having a particular attribute, cells are generally seeded onto a clean sterile support substrate or layer 72 and allowed to proliferate in an environment which is conducive to cell growth. The seeding of cells may be carried out with a particular number of cells or particular cell densities being seeded or otherwise transferred and may be transferred from existing cultures or from primary sources. Cell counts of about 100 cells to about 100,000,000 cells may be transferred or otherwise plated for some embodiments, and, depending on the area occupied by the cells being transferred, which may be in confluence, semi-confluence or non-confluent. Cell densities and determination of confluence may be carried out by any suitable method such as the use of a reticle in the field of view of a microscope, a hemocytometer or the like. With regard to cell passaging, the term as used herein in a general sense is meant to encompass the separation of cells from other cells and exposing the separated cells to new conditions, which may include seeding the separated cells onto a new substrate.
The cell line shown includes an exemplary human stem cell line which has spread across the top surface of the support layer 72 and has produced zones of differentiated cells 77 and undifferentiated cells 79. For stem cells, the cells tend to stay together or in proximity to adjacent cells, but may begin to differentiate into specific cell types over time, and particularly if the cells become too crowded as a result of cell proliferation. For many applications, it may be desirable to cultivate only the undifferentiated cells and so only those will be transferred to a new cell culture dish 74 for further proliferation. It is the ability to partition a cell culture layer 70 as shown in
As discussed above, during such a pass of the roller body 12, it may be desirable for the layer penetrating structure 16 to roll over and partition the cell culture layer 70 without sliding or skidding on the cell culture layer 70 or the support substrate 72 in order to avoid cross contamination or deformation of either of the layers. In addition, it may be desirable for the layer penetrating structure 16 to contact the cell culture layer 70 and be pulled away from the cell culture layer 70 during the partitioning process without picking up cells or otherwise having cells of the cell culture layer 70 adhere to the layer penetrating structure 16. This may be achieved in some embodiments by having a cell culture layer 70 with particular surface properties and a layer penetrating structure 16 with an outer surface that is smooth and non-adherent.
Once the first pass is completed, the process may be repeated as many times as necessary by repositioning the roller body 12 adjacent to the partition lines 78 of the first pass and passing the roller body 12 across the cell culture layer 70 adjacent and parallel the first pass to produce a second set of partition lines 78 adjacent and parallel the first. The process may be repeated until the entire surface, or most of the surface, of the cell culture layer 70 has been partitioned into elongate parallel strips 82 with the partitions 78 extending at least from a top surface 84 of the cell culture layer 70 to a bottom surface of the cell culture layer 70 as shown in
Once this set of partitions 78 has been produced, a second set of partition lines 78 or furrows, which may be substantially perpendicular to the first set of partition lines 78 for some embodiments, are produced in the same way as described above with the same cell culture layer processing tool 10 as shown in
Once the cell culture layer 70 or a desired portion thereof has been partitioned into isolated divided portions 88, each isolated divided portion having substantially the same surface area dimensions, the divided portions 88 may then be removed from the cell culture dish 74 and support layer 72 for any desired purpose by a variety of methods. The isolated divided portions 88 of the cell culture layer to be removed may be selected for any desired attribute or characteristic, such as any of the attributes or characteristics of cells discussed above. For example, it may be desirable to remove and transport all isolated divided portions 88 of the cell culture layer having cells in a desired state, such as a differentiated state or undifferentiated state.
The divided portion 88 may also be transferred to a tube (not shown) or other suitable vessel for spin down as well as other processing. For example, in some embodiments, the divided portion 88 may be transferred to a solution in a 15 ml tube for spin down at about 800 to about 1200 rpm for about 1.5 minutes to about 2.5 minutes. The solution in the tube may also be left to stand stationary over a period of time suitable for the cells in the solution to settle to the bottom of the tube by gravity. The cells may then be returned singly and re-suspended in a new growth medium and transferred to a new support layer. Once transferred and seeded, the newly seeded cell culture dish may be allowed to proliferate with at least one heating and cooling cycle or other environmental support that is conducive to cell growth and multiplication. For some embodiments, multiple heating and cooling cycles may be used to promote cell growth. In this way, the desirable features of the initial cell line used to seed the initial cell culture dish 74 may be cultivated and expanded to produce a high volume of cells from the line with desirable traits. It may also be desirable to perform some or all of the procedures discussed above under a laminar flow hood or in another type of suitable purified environment to avoid contamination of the cell culture.
A schematic illustration of a cell culture layer 70 of human stem cells is shown in
Although the cell layer processing tool embodiments described above have been primarily directed to a hand operated configurations, it may be desirable for some applications to use the same or similar roller body 12 embodiments in an automated format.
A support structure 108 is coupled to the roller body 12 and is configured to allow rotation of the roller body 12 about an axis that is substantially concentric with the cylindrical outer surface 14 of the roller body 12. The support structure 108 is secured to a movable carrier 110 of the three axis robotic positioning actuator 104. With this arrangement, the controller 106 may include a processor (not shown) that may be programmed to move the moveable carrier 110 in a controllable manner in each of three orthogonal axes so as to controllably position and move the roller body 12. In this way, the roller body 12 and layer penetrating structure 16 thereof may be positioned and moved across a cell culture layer 70 in the same way as the roller body embodiments discussed above. This allows a cell culture layer 70 to be partitioned into divided portions 88 as discussed above in a repeatable manner without the need for a human operator and thus reducing human/operator error.
A cell culture dish 74 having a cell culture layer 70 disposed on a support layer 72 in the cell culture dish 74 may be placed under the roller body 12 which is coupled to the moveable carrier 110 by the support structure 108. The cell culture dish 74 may be fixed in the x-y plane position by a plurality of stops 112 that are configured to hold the cell culture dish 74 in place by spacing them around a circular radius that is just slightly larger than an outer radius of the pre-selected cell culture dish 74. The movable carrier 110 and roller body 12 coupled thereto may then be lowered into contact with the cell culture layer 70 and moved across the cell culture layer 70 with a substantially constant downward force to partition the cell culture layer 70 into divided portions 88. The moveable carrier 110 may be actuated by a servo motor or the like corresponding to each axis of the three axis positioning actuator 104. There may be a servo motor or the like configured to move the moveable carrier 110 in a z-axis direction as indicated by arrows 114, in the y-axis direction as indicated by arrows 116 and in the x-axis direction as indicated by arrows 118.
For some embodiments of cell culture processing tools, a method of use may include specific procedures in order to obtain desired results. The following discussion includes a series of exemplary procedures for use of a cell culture processing tool, such as cell culture processing tool 10 or any of the other cell culture processing tools, or components thereof, discussed above. For some such embodiments, the processing tool represents a novel human embryonic stem cell passaging device that makes manual passaging of stem cell colonies more rapid and reproducible. The processing tool is made of a cell-culture safe inert material that facilitates cutting of the embryonic stem cell colonies growing on feeder cells or other substrate into uniform sized pieces for reproducible and optimal passaging. This tool outperforms existing manual and enzymatic passing methods in speed, uniformity of passaged colonies, and reliability and should be stored at room temperature.
Some advantages of the cell culture processing tool may include manual passaging stem cell colonies in a fraction of the time compared with standard techniques, stem cell colonies are cut in pieces of uniform size, making passaging of stem cells more reproducible and the processing tool is cell-culture safe, ready for-use and packaged individually in gamma-irradiated sleeves. During use, procedures often are performed aseptically under a laminar flow hood. This embodiment of the cell culture processing tool is intended for one-time use and users generally do not sterilize (alcohol or autoclave) the processing tool, since the tool may lose shape and cease to function properly.
In use, the differentiated portions of human embryonic stem cell culture may optionally be dissected out using a poker or spike and thereafter removed by changing the medium. In general, the processing tool is used by first pulling open packaging and removing the processing tool 10 under a laminar flow hood. A culture vessel is held in one hand and the processing tool pulled across the entire plate in one direction as shown in
With regard to the above detailed description, like reference numerals used therein refer to like elements that may have the same or similar dimensions, materials and configurations. While particular forms of embodiments have been illustrated and described, it will be apparent that various modifications can be made without departing from the spirit and scope of the embodiments of the invention. For example, any of the roller body embodiments 12, 48 and 54 may be used in conjunction with handle 22 or the robotic cell culture processing tool 102. Accordingly, it is not intended that the invention be limited by the forgoing detailed description.
This application is a continuation application of U.S. application Ser. No. 12/180,473, filed Jul. 25, 2008, and claims priority to U.S. application No. 60/953,896, filed Aug. 3, 2007, which disclosures are herein incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60953896 | Aug 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12180473 | Jul 2008 | US |
| Child | 13526350 | US |
