Patent Application
20080200345
The present invention relates generally to the field of high throughput screening methods. In particular, the present invention relates to high throughput screening methods that can be used to identify mixtures of single agents and single agents within these mixtures that elicit a desired biological response in the cell.
It is known that attachment-dependent cells need a suitable culture substrate that allows for cell attachment in order to survive in vitro cell culture. Typically, proteins in media immobilize arbitrarily onto the surface of the cell culture substrate to form a layer to which cells can attach. The cell surface receptors, e.g., integrins, mediate cell attachment to such a protein layer, for example, by reacting with an extracellular matrix (ECM) protein such as fibronectin, that is present and still biologically active in this serum protein layer. Upon cell attachment to a surface through cell surface receptor-ligand interactions, internal signaling pathways are triggered within the cell, ultimately determining the fate of the cell, e.g., survival, proliferation, or differentiation. A disadvantage of using serum protein contained in the media to attach cells to the cell culture substrate is that, in contrast to in vivo biological processes, signaling pathways are triggered non-specifically and arbitrarily due to the non-specific and arbitrarily formed serum protein layer. Another disadvantage is that protein that is adsorbed onto the substrate from the media can be solubilized back into the media, and thus leave the surface, which further results in the substrate surface being poorly defined.
In other conventional cell culture systems, proteins to which cells can attach can be in the form of protein coatings that have been applied to the culture vessel prior to adding cells in cell culture media. Proteins that are adsorbed as the coating on the culture surface can be solubilized back into the culture medium and thus leave the culture surface.
For cells to be used in therapies to treat or cure diseases in humans, it is desirable to control cell fate, e.g., cell survival, proliferation and differentiation, when cells are maintained in culture in vitro. It is thus necessary to control cell surface receptor interaction with ligands present on the in vitro culture substrate. In order to gain control over cell-surface interactions, a suitable culture substrate, such as polystyrene, can be coated with a polymer which does not allow for cell attachment, even when serum proteins are used in the culture media. This coating thus eliminates the uncontrolled and arbitrary adsorption of the serum proteins. Biologically active ligands suitable to interact with cell surface receptors are then immobilized on this coating while maintaining the biological activity of the ligands. This concept is known. For example, it is known to use hyaluronic acid or algenic acid as a surface coating upon which the cell adhesion ligands can be immobilized using chemistries resulting in stable covalent bonds between the coating and the cell adhesion ligands. This prevents the cell adhesion ligand from being solubilized and leaving the surface. Moreover, the coating itself does not support cell adhesion. This is described in copending, commonly owned U.S. application Ser. No. 10/259,797, filed Sep. 30, 2002.
It is known to study one immobilized ligand and its effect on a certain cell type at a time. However, it is likely that mixtures of cell adhesion ligands and extrinsic factors are required in order to achieve a desired cell fate. A great number of cell adhesion ligands are known and used in cell adhesion studies. It can thus be a tedious task to find the right cell adhesion ligand or cell adhesion ligand combinations to place on a cell culture surface for optimal cell adhesion for a given cell type.
Therefore, there is a need in the art for higher throughput methods to identify cell adhesion ligands and/or extrinsic factors for a given cell type. This is of particular interest for cells that do not survive or only survive by drastically altering their differentiation state in conventional cell culture systems, a prime example being primary mammalian cells. In particular, there is a need in the art for a statistical experimental design that can be used to systematically explore the interactions between mixtures of factors that are required in order to achieve a desired fate for a given cell type.
The present invention provides a high throughput method for identifying agents capable of producing a desired biological response in whole cells. In particular, the method includes the steps of providing receptacles having a culture surface; placing different mixtures of single agents into selective ones of the receptacles according to a statistical design; and immobilizing the mixtures of single agents to the culture surface. The method further includes contacting the immobilized agents with the whole cells; and acquiring data which is indicative of a desired biological response in the contacted cells. The method also includes using statistical modeling of the acquired data to determine which mixtures of single agents and/or which single agents in these mixtures are effective in producing the desired biological response in the contacted cells.
As defined herein, “agents” are growth effector molecules that bind to cells and regulate the survival, differentiation, proliferation or maturation of target cells or tissue. Examples of suitable agents for use in the present invention include growth factors, extracellular matrix molecules, peptides, hormones and cytokines.
The term “agent-immobilizing material” is defined herein as a biocompatible polymer that can serve as a link between the culture surface and an agent.
As defined herein, the term “immobilize,” “immobilized,” and the like is to render an agent(s), i.e., growth effector molecules, immobile on a culture surface, such as a well surface or the surface of a scaffold contained within a well. This term is intended to encompass passive adsorption of the agents to the culture surface, as well as direct or indirect covalent attachment of the agents to the culture surface.
“Factors” are the names of the variables in the experiment, and represent the things that the experiment changes from one trial or run (for e.g., one well) to the next. In the present invention, “factor” is a generic name for a single agent or mixture of single agents. Factors are combined according to a statistical design to form different mixtures in the experiment.
“Statistical Design”, as defined herein is an experimental design that assists the user in finding a combination of adjustable variables (i.e., factors) to produce the best experimental outcome, dramatically reducing the number of experiments needed to achieve that objective. In the present invention, a suitable statistical design is generated using generic factor names which represent the agents being tested. The design includes factor levels that can be the amounts and/or concentrations of the factors or that can be converted to the actual amounts and/or concentrations of the factors. The design also includes experimental runs, which are numbered. Experimental runs specify the combinations of factors and the levels thereof to test, and each corresponds to a single well on a multiwell plate, for example. The experimental runs can be mapped to wells on a generic multiwell plate.
As used herein, the terms “pre-treatment” and “pre-treated” refers to the addition to a surface or other substrate of functional groups which are chemically involved in the covalent bond subsequently formed with the agent-immobilizing material (i.e., a biocompatible polymer). For example, a surface of a microtitre well can be subjected to amino-plasma treatment to create an amine-rich surface onto which the agent-immobilizing material may be coupled.
As described above, the present invention relates to a high throughput method for identifying agents capable of producing a desired biological response in whole cells. This method includes the steps of: providing receptacles having a culture surface; placing different mixtures of single agents into selective ones of the receptacles according to a statistical design; immobilizing the mixtures of single agents to the culture surface; and contacting the immobilized agents with the whole cells. The method further includes acquiring data indicative of the desired biological response in the contacted cells; and determining which mixtures of the single agents and/or which of the single agents in these mixtures are effective in producing the desired biological response using statistical modeling of the acquired data. In one desired embodiment, the desired biological response may be selected from the following: cell adhesion, cell survival, cell differentiation, cell maturation, cell proliferation and combinations thereof.
As described above, it is likely that mixtures of single agents are required in order to achieve a desired cell fate. A great number of growth effectors are known. For example, growth effector molecules that bind to cell surface receptors or are taken up through ion channels or transports and regulate the survival, differentiation, proliferation or maturation of these cells include growth factors, extracellular matrix molecules, peptides, hormones and cytokines, of which there are many examples. It can therefore be a tedious task to find the right growth effector or growth effector combinations to place on a cell culture surface to achieve a desired cell fate for a given cell type.
The present invention solves a need in the art by providing for higher throughput methods to identify mixtures of agents that elicit a desired biological response for a given cell type.
In preferred embodiments of the method of the present invention, mixtures of single agents are covalently immobilized to an agent-immobilizing material on a culture surface, such as the receptacle surface. It is also well within the contemplation of the present invention that mixtures of single agents can be passively adsorbed onto the culture surface. The culture surface to which the agents are immobilized can also be a scaffold contained within the receptacle.
Referring now to
With reference now to
Referring now to
For example, as shown in
With reference now to
As shown schematically in
With reference now to
It is noted that each of the scenarios depicted in
The present invention provides for methods which use a format, such as a 96-well plate format, to screen a plurality of different mixtures of agents in parallel for their ability to elicit a desired response in a cell. In one embodiment, the method involves placing different mixtures of agents into selective wells of a multi-well plate according to a statistical design. The method may further include the step of placing single agents into other of the wells. The agents are subsequently immobilized to a culture surface, such as a well surface. The method also includes delivering a fluid sample comprising a cell-type to the wells. After an appropriate incubation time between the cells and the samples in the various wells, evidence of an interaction between the cells and the well components can be detected, either directly or indirectly. For example, data can be acquired using functional assays, immunocytochemistry, or microscopy.
Suitable statistical designs for use with the present invention include, but are not limited, to the following: fractional factorial design, D-optimal design, mixture design and Plackett-Burman design. In one preferred embodiment, the statistical design is a mixture design. In another embodiment, the design is a space-filling design based on a coverage criteria, a lattice design, or a latin square design.
In desired embodiments, the culture surface, which may be pre-treated, is coated with an agent-immobilizing material. The agent-immobilizing material is desirably a biocompatible polymer which does not support cell adhesion and which can serve as a flexible link (tether) between the culture surface and the agents. Examples of suitable polymers include synthetic polymers like polyethylene oxide (PEO), polyvinyl alcohol, polyhydroxylethyl methacrylate, polyacrylamide, and natural polymers such as hyaluronic acid and algenic acid.
In desired embodiments, culture surfaces are selected from, but not limited to, the following: polystyrenes, polyethylene vinyl acetates, polypropylene, polymethacrylate, polyacrylates, polyethylenes, polyethylene oxide, glass, polysilicates, polycarbonates, polytetrafluoroethylene, fluorocarbons, and nylon. It is also well within the contemplation of the present invention that the culture substrates may wholly or partially include biodegradable materials such as polyanhydrides, polyglycolic acid, polyhydroxy acids such as polylactic acid, polyglycolic acid and polylactic acid-glycolic acid copolymers, polyorthoesters, polyhydroxybutyrate, polyphosphazenes, polypropyl fumurate, and biodegradable polyurethanes.
The culture surface to which the agents can be adsorbed or tethered can be pre-treated. For example, cell culture surfaces bearing primary amines can be prepared by plasma discharge treatment of polymers in an ammonia environment. The agent-immobilizing material can then be covalently attached to these aminated surfaces using standard immobilization chemistries, as described in copending, commonly owned U.S. application Ser. No. 10/259,797, filed Sep. 30, 2002, the entire contents of which are incorporated herein by reference. Two processes used commercially to create tissue culture treated polystyrene are atmospheric plasma treatment (also known as corona discharge) and vacuum plasma treatment, each of which is well known in the art. Plasmas are highly reactive mixtures of gaseous ions and free radicals. An amino-plasma treatment or oxygen/nitrogen plasma treatment can be used to create an amine-rich surface onto which biocompatible polymers such as hyaluronic acid (HA) or algenic acid (AA) may be coupled through carboxyl-groups using carbodiimide bioconjugate chemistries, as described in U.S. application Ser. No. 10/259,797. The resulting surfaces will not allow cells to attach, even in the presence of high, e.g., 10-20% serum protein concentrations present in the cell culture media. An example of pre-treated tissue culture polystyrene products that can be used to covalently link the agent via the agent-immobilizing material are the PRIMARIA™ tissue culture products (Becton Dickinson Labware), which are created using oxygen-nitrogen plasma treatment of polystyrene and which result in the incorporation of oxygen- and nitrogen-containing functional groups, such as amino and amide groups.
Agents, such as extracellular matrix proteins, peptides, etc. can be subsequently covalently coupled to the HA or AA surface described above utilizing the amine groups on the proteins/peptides and either the carboxyl groups on the HA or AA, or aldehyde groups created on the HA or AA by oxidation using sodium periodate, for example.
For example, the terminal sugar of human placental hyaluronic acid can be activated by the periodate procedure described in E. Junowicz and S. Charm, “The Derivatization of Oxidized Polysaccharides for Protein Immobilization and Affinity Chromotography,” Biochimica et. Biophysica Acta, Vol. 428: 157-165 (1976), incorporated herein by reference. This procedure entails adding sodium or potassium periodate to a solution of hyaluranic acid, thus activating the terminal sugar which can be chemically cross-linked to a free amino group on an agent, such as the terminal amino group on an extracellular matrix protein. In another preferred embodiment, free carboxyl groups on the biocompatible polymer (for example, HA or AA) may be chemically cross-linked to a free amino group on the agent using carbodiimide as a cross-linker agent. Other standard immobilization chemistries are known by those of skill in the art and can be used to join the culture surfaces to the biocompatible polymers and to join the biocompatible polymers to the agents. For example, see “Protein Immobilization: Fundamentals and Applications” Richard F. Taylor, Ed. (M. Dekker, NY, 1991) or copending U.S. application Ser. No. 10/259,797, filed Sep. 30, 2002.
It is noted that whereas the tethering of the agents to aminated tissue culture surfaces via biocompatible polymers comprises one embodiment of the present invention, these agents can also be tethered via biocompatible polymers to carboxylated surfaces or hydroxylated surfaces using standard immobilization chemistries. Examples of attachment agents are cyanogen bromide, succinimide, aldehydes, tosyl chloride, avidin-biotin, photocrosslinkable agents, epoxides and maleimides.
As described above, it is an aspect of the present invention that mixtures of agents are contained within selective ones of the receptacles. Moreover, it is a further aspect of the present invention that other receptacles may contain a single agent. These agents may be tethered alone or in combinations to pre-treated tissue culture surfaces. The agents may be combined in any desired proportions. The relative amounts of different agents present on the culture surfaces can be controlled, for example, by the concentration of the agents in a coating composition. Alternatively, the loading density can be controlled by adjusting the capacity of the biocompatible polymers bound to the culture surface. This can be accomplished by, for example, controlling the number of reactive groups on the polymers that can react with the agents or by controlling the density of the biocompatible polymer molecules on the culture surface. Moreover, the agents can first be separately linked to the biocompatible polymers (tethers), and then the “loaded” tethers can be mixed in the desired proportions, and attached to the pre-treated substrate.
As described above, it is preferred that the agents are covalently immobilized via biocompatible polymers to a pre-treated tissue culture surface, which is desirably amine-rich. However, it is noted that it is well within the contemplation of the present invention that rather than covalently immobilizing the agents to the surfaces in this way, the agents can be immobilized to the culture surface (e.g., well surface) by passively adsorbing the agents to the surface. It is also well within the contemplation of the present invention that the agents can be immobilized on or impregnated within a scaffold, which can be placed in the receptacle and contacted with fluid containing the cells. Suitable scaffolds for use in the present invention and methods for immobilizing agents thereto or therewithin are described in copending, commonly owned U.S. application Ser. No. 10/259,817, filed Sep. 30, 2002, the entire contents of which are incorporated herein by reference.
Receptacles for use in the present invention can take any usual form, but are desirably tissue culture dishes, multi-well plates, flasks, tubes, and roller bottles. Configurations such as microtitre wells and tubes are particularly useful in the present invention and allow the simultaneous assay of a large number of samples to be performed manually in an efficient and convenient way. The assay can also be automated using, for example, microtitre wells and is capable of extensive automation because of automatic pipetters and plate readers. Other solid phases, particularly other plastic solid supports, may also be used.
It is noted that the method steps of the present invention can be readily automated. This is particularly so with microtitre plates as the format. Therefore, in one embodiment of the present invention, the receptacles can comprise the wells of a 96-well microtitre plate. Automatic pipetting equipment (for reagent addition and washing steps) and color readers already exist for microtitre plates. An example of an automated device for carrying out the present invention can include: a pipetting station and a detection apparatus, the pipetting station being capable of performing sequential operations of adding and removing reagents to the wells at specific time points in a thermostatic environment (i.e., temperature controlled environment).
As described above, agents for use in the present invention are growth effector molecules that bind receptors on the cell surface or are taken up through ion channels or transports and regulate the growth, replication or differentiation of target cells or tissue. In one embodiment, these agents are cell adhesion ligands and/or extrinsic factors. In desired embodiments, the agents can be extracellular matrix proteins, extracellular matrix protein fragments, peptides, growth factors, cytokines, and combinations thereof.
Preferred agents are growth factors and extracellular matrix molecules. Examples of growth factors include, but are not limited to, vascular endothelial-derived growth factor (VEGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF.alpha., TGF.beta.), hepatocyte growth factor, heparin binding factor, insulin-like growth factor I or II, fibroblast growth factor, erythropoietin nerve growth factor, bone morphogenic proteins, muscle morphogenic proteins, and other factors known to those skilled in the art. Other suitable growth factors are described in “Peptide Growth Factors and Their Receptors I” M. B. Sporn and A. B. Roberts, Eds. (Springer-Verlag, NY, 1990), for example.
Growth factors can be isolated from tissues using methods known in the art. For example, growth factors can be isolated from tissue or can be produced by recombinant means. For example, EGF can be isolated from the submaxillary glands of mice and Genentech (South San Francisco, Calif.) produces TGF-.beta. recombinantly. Other growth factors are also available from vendors, such as Sigma Chemical Co. (St. Louis, Mo.), R&D Systems (Minneapolis, Minn.), BD Biosciences (San Jose, Calif.), and Invitrogen Corporation (Carlsbad, Calif.), in both natural and recombinant forms.
Examples of suitable extracellular matrix molecules for use in the present invention include vitronectin, tenascin, thrombospondin, fibronectin, laminin, collagens, and proteoglycans. Other extracellular matrix molecules are described in Kleinman et al., “Use of Extracellular Matrix Components for Cell Culture,” Analytical Biochemistry 166: 1-13 (1987), or known to those skilled in the art.
Additional agents useful in the present invention include cytokines, such as the interleukins and GM-colony stimulating factor, and hormones, such as insulin. These are described in the literature and are commercially available.
Cells for use with the present invention can be any cells that can potentially respond to the agents or that need the agents for growth. For example, cells can be obtained from established cells lines or separated from isolated tissue. Suitable cells include most epithelial and endothelial cell types, for example, parenchymal cells, such as hepatocytes, pancreatic islet cells, fibroblasts, chondrocytes, osteoblasts, exocrine cells, cells of intestinal origin, bile duct cells, parathyroid cells, thyroid cells, cells of the adrenal-hypothalamic-pituitary access, heart muscle cells, kidney epithelial cells, kidney tubular cells, kidney basement membrane cells, nerve cells, blood vessel cells, cells forming bone and cartilage, and smooth and skeletal muscles. Other useful cells can include stem cells which may undergo a change in phenotypes in response to a select mixture of agents. Further suitable cells include blood cells, umbilical cord blood-derived cells, umbilical cord blood-derived stem cells, umbilical cord blood-derived progenitor cells, umbilical cord-derived cells, placenta-derived cells, bone marrow-derived cells, and cells from amniotic fluid. The cells can be genetically engineered. In preferred embodiments, the cells are cultured with agents which are tethered via a biocompatible polymer to a culture substrate, such as a well surface(s) of a 96-well microtitre plate. These cells can be cultured using any of the numerous well known cell culture techniques, such as those described in Freshney, “Cell Culture, A Manual of Basic Technique” 3.sup.rd Edition (Wiley-Liss, NY, 1994). Other cell culture media and techniques are well known to those skilled in the art and can be used in the present invention.
Statistically designed experiments in accordance with the present invention will now be described.
An oxygen/nitrogen plasma is used by Becton Dickinson Labware to create PRIMARIA™ tissue culture products. In particular, oxygen/nitrogen plasma treatment of polystyrene products results in incorporation of oxygen- and nitrogen-containing functional groups, such as amino and amide groups. For this experiment, HA was coupled to the amine-rich surface on PRIMARIA™ multi-well plates through carboxyl groups on HA using carbodiimide bioconjugates chemistries well known in the art, such as those described in “Protein Immobilization: Fundamentals and Applications” Richard S. Taylor, Ed. (M. Dekker, NY, 1991) or as described in copending U.S. application Ser. No. 10/259,797, filed Sep. 30, 2002.
ECM agents were covalently attached to the HA polymer tethered to the culture surface. In particular, aldehyde groups were created on HA by oxidation using the periodate procedure described in E. Junowicz and S. Charm, “The Derivatization of Oxidized Polysaccharides for Protein Immobilization and Affinity Chromotography,” Biochimica et. Biophysica Acta, Vol. 428: 157-165 (1976). This procedure entailed adding sodium periodate to a solution of HA, thus activating the terminal sugar. Subsequently, the activated HA was coupled to the amine groups on the ECM proteins using standard immobilization chemistries, such as those described in “Protein Immobilization: Fundamentals and Applications” Richard F. Taylor, Ed. (M. Dekker, NY, 1991) or copending U.S. application Ser. No. 10/259,797, filed Sep. 30, 2002.
In the present example, the statistical design is a mixture design. This design was used to identify pairs of factors, or single factors that had a positive effect on a cell response, and allows us to look at interactions between two ECMs. In this example, 10 single ECMs, each representing a single “factor” are used to created ECM mixtures for placement into the wells of a 96-well plate as shown in
In this example, a group of 10 adhesion ligands was selected and a 96-well plate was chosen as the format for this screen. To eliminate border effects due to uneven evaporation, only the inner 60 wells of the 96-well plate are to be used for the experiment. Wells in the outer rows and columns of the plate can thus be used for suitable controls.
The following 10 adhesion ligands were selected based on their common use as cell culture reagents, commercial availability and price: Collagen I (CI), Collagen III (CIII), Collagen IV (CIV), Collagen VI (CVI), elastin (ELA), fibronectin (FN), vitronectin (VN), laminin (LAM), polylysine (PL), and polyornithine (PO).
A statistical design was developed with special consideration of the surface chemistry requirements. In particular, in this experiment the scenario shown in
With reference now to
MC3T3-E1 cells, originated from Dr. L. D. Quarles, Duke University, and were kindly provided by Dr. Gale Lester, University of North Carolina at Chapel Hill. These cells were grown using standard cell culture techniques. MC3T3-E1 is a well-characterized and rapidly growing osteoblast cell line that was chosen because it attaches aggressively to most commonly used tissue culture surfaces.
Cells were removed from cell culture flasks using trypsin-EDTA according to methods well known in the art. Cells were enumerated, spun down and resuspended in media containing no serum or, alternatively, in media containing 10% fetal calf serum. Cells were plated into the wells of a 96-well plate according to the layout shown in
An image analysis software package (Meta Morph, Universal Imaging Corporation, a subsidiary of Molecular Devices, Downingtown, Pa.) was used to enumerate the fluorescently labeled cell nuclei in
In
In order to optimize the surfaces, one can follow two leads, e.g., the “best well” composition or the “best factors”. The determination of “best factors” is made following rigorous statistical analysis of the experimental results.
In the “best well” approach, the well with the best experimental outcome is chosen for further optimization. In the example shown in
In the “best factors” approach, the experimental results are analyzed using statistical models. For the above-described example, a mixture-model analysis of the MC3T3-E1 data shows that Collagen IV, laminin, and poly-L-lysine (marginal effect) appear to increase the cell count when present at significant quantities with no serum as shown in
With reference now to
It is noted that both the “best well” and “best factors” approaches are valid, but each approach can lead to different surface compositions. In the present example, the “best well” approach would lead to a surface comprising Collagen-type VI and Collagen-type III, while the “best factor” approach would lead to a surface comprising Collagen VI and laminin.
Design
The present example describes a Plackett-Burman (PB) design as shown in
Proposed Acquisition of Data and Statistical Analysis
Cells are plated into the wells of a 96-well plate in accordance with the design shown in the spreadsheet of
Following the first screen, the main effects are estimated and reviewed. By “main effects”, it is meant the effect of a single agent acting independently. Interaction effects mean the combined effects of more than one single agent when the agents act in concert (not independently). At this point, relevant interactions among the agents typically are not estimated in the statistical model, but interactions among the agents would be expected to result in the best experimental runs, i.e., best wells. After the first round of screening, the best wells and the factors that are included in these wells (level=“1”) are identified. Follow-up experiments can be performed for each best well using all the factors included in the well, whether or not they had a positive, neutral, or negative effect in the preliminary statistical analysis. The experiments can be repeated with a subset of the agents identified in the best well so as to arrive at an optimum subset of factors for producing a desired response in a cell. Moreover, the experiment can be repeated, wherein the concentration of the agents in a best well are varied. Follow-up experiments can also be performed with the subset of single agents that had statistically significant main effects or by combining a subset of the best single agents with a subset of agents identified in the best mixtures.
It has been proposed that the control of cellular phenotypes via extracellular conditions is governed by high order interactions among the factors in the extracellular environment. The Plackett-Burman design presented here is believed to provide good statistical estimates of the main effects and also provides the opportunity to observe a diverse set of combinations of factors among its experimental runs. In this case, higher-order interactions would be expected to result in specific experimental runs as being “best wells” over and above what could be predicted by the individual main effects of the agents in the best wells.
This application is a Divisional application of application Ser. No. 10/662,640, filed Sep. 15, 2003, the contents of which are incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10662640 | Sep 2003 | US |
| Child | 12015233 | US |
