Patent Application
20140329319
The present invention relates generally to apparatus and method for polymer modification of biological cells, and more particularly relates to the immunocamouflage of biological cells.
Polymers such as methoxypolyethylene glycol (mPEG), hyperbranched polyglycerols (HPG) and polyoxasolines (POZ) are known for their non-toxic properties. In the field of immunology, these polymer classes are particularly useful for improving biocompatibility and reducing immunological recognition of cells when it is covalently grafted to cell surfaces.
Most common techniques for grafting polymers to biological cells involve manually mixing a biological cell solution and an activated polymer in buffer and then agitating the resulting mixture to obtain polymer-modified cells. This manual (i.e. non-automated) mixing method usually results in a high hydrolysis rate of activated polymer, poor homogeneity of polymer-modified cells product, low control of the mixture's sterility, in addition to being relatively slow, time consuming and unsuitable for scaling up to process larger volumes.
Accordingly there remains a need for an improved device and method for grafting polymers to biological cells.
In accordance with one aspect of the present invention, there is provided a device for grafting polymers to biological cells to immunocamouflage the biological cells comprising: a first reservoir containing a first liquid mixture of biological cells; a second reservoir containing a second liquid mixture of activated polymer; at least one pump in fluid flow communication with both the first and second reservoirs and which respectively transfers the first and second liquid mixtures from the first and second reservoirs into a common mixing chamber, the pump being operable to independently control an output volume of each of the first and second liquid mixtures fed from each of the first and second reservoirs into the mixing chamber; the mixing chamber being in fluid flow communication with the at least one pump and receiving therein said output volumes of the first and second liquid mixtures, the output volumes mixing within the mixing chamber in a predetermined volume ratio to produce a final mixture comprising polymer-modified biological cells; and an outlet for evacuating the final mixture of said polymer-modified biological cells from the mixing chamber.
There is also provided, in accordance with another aspect of the present invention, a method for producing polymer-modified biological cells comprising the steps of: pumping a first liquid mixture comprising biological cells from a first reservoir to a mixing chamber and pumping a second liquid mixture comprising activated polymer from a second reservoir to said mixing chamber; independently controlling a predetermined output volume of the first and second liquid mixtures pumped into the mixing chamber; mixing the predetermined output volumes of the first and second liquid mixtures in the mixing chamber to produce a final mixture ratio comprising polymer-modified biological cells; and evacuating the final mixture comprising the polymer-modified biological cells from the mixing chamber into an output reservoir.
Further features and advantages will become apparent from the following detailed description, taken in combination with the appended drawings, in which:
The present disclosure relates generally to devices and methods for grafting polymers to biological cells such as to produce biological cells having covalently grafted polymers (i.e. “polymer-grafted” or “polymer-modified” cells), a process which is referred to as immunocamouflage of the biological cells. The process for grafting polymers to biological cells requires a biological cell solution to be mixed with an activated polymer, such as to subsequently obtain polymer-modified cells. The activated polymers used in conjunction with the devices and methods of the present disclosure may comprise, but are not limited to, methoxypolyethylene glycol (mPEG), hyperbranched polyglycerol and/or polyoxasolines. These activated polymers have chemical linker groups and may comprise a number of different activation chemistries. Linker molecules may comprise, inter alia, cyanuric chloride, imidazolyl formate, succinimidyl succinate, succinimidyl carbonate, succinimidyl glutarate, N-hydroxysuccinimide, 4-nitrophenol, and 2,4,5-trichlorophenol. The linker molecules listed above are exemplary only. Any linker molecule capable of covalently attaching to the polymer may be similarly used.
The devices and systems for the immunocamouflage of biological cells of the present disclosure comprise generally a first reservoir containing a first liquid mixture of biological cells and a second reservoir comprising a second liquid mixture of one or more activated polymer (as exemplified by methoxypolyethylene glycol (mPEG), hyperbranched polyglycerol or polyoxasolines, for example). The first and second liquid reservoirs are in fluid communication with at least one fluid transfer device in the form of a pump which may have a variable and controllable output. The pump(s) respectively transfer(s) the first and second mixtures from the first and second reservoirs into a common mixing chamber. The fluid transfer device/pump independently controls at least one flow characteristic from each of the first and second reservoirs, including output volume, mass flow, flow rate, etc. The pump(s) thereby controls, for example, the volume of the first and second liquid mixtures received within the mixing chamber, such that these mixtures are mixed at a predetermined volume ratio. By mixing the first and second liquid mixtures at a predetermined volume ratio, a more homogenous and reproducible final mixture of polymer-modified biological cells is obtained. An outlet for evacuating the final mixture, comprising the polymer-modified biological cells, from the mixing chamber may also be provided.
Referring to
In accordance with one embodiment, the ratio of biological cells and activated polymer micro-mixing in the mixing chamber 18 is controlled by the volumes of the first and second liquid mixtures pumped by the syringes 14, 16 at fixed concentrations.
The syringes 14, 16 may therefore be configured to pump a substantially equal volume of the first and second liquid mixtures. For example, 60 mL syringes can be used for each syringe 14, 16. Alternatively, different sizes of syringes may be used but each would be filled with a substantially equal volume of the first and second liquid mixtures.
In another embodiment, the output volume of the first and second liquid mixtures may be different and can be controlled by using different syringe volumes or different volumes of liquid mixtures within the syringes of the same size.
Alternatively still, as shown in
It is known that when a solution of chemically activated polymers is prepared in a large volume, a high hydrolysis rate of the activation chemistry is observed, which results in an inactivation of the polymer and therefore reduced time-dependent covalent grafting to biological cells resulting in a lower homogeneity of polymer grafted biological cells. The embodiment illustrated in
The device 10 further comprises an outlet conduit 32 in fluid communication with the mixing chamber 18 for evacuating the final mixture comprising polymer-modified biological cells from the mixing chamber 18. As illustrated in
Referring now to the alternate embodiment of
While it may be possible to directly connect the peristaltic pumps 112, 113 to the first and second reservoirs 114, 116, in the depicted embodiment of the device 110 a first tube 120 is in fluid communication with the first reservoir 114 and the mixing chamber 118 and a second tube 122 is in fluid communication with the second reservoir 116 and the mixing chamber 118, with the first peristaltic pump 112 being in-line with the first tube 120 and the second peristaltic pump 113 being in-line with the second tube 130. In alternative embodiments, other piping and/or tubing configurations may be employed, provided that the peristaltic pumps 112, 113 are disposed in-line between each of the reservoirs 114, 116 and the common mixing chamber 118.
The device 110 for the immunocamouflage of biological cells by polymer grafting of
In one possible embodiment of the device 110, one or more additional peristaltic pumps are provided to feed the solution into the first and second reservoirs 114, 116. These may be provided, for example, in line between the external reservoirs 128 and 130 and the first reservoir 114 (either a single peristaltic pump for both external reservoir feeds or one for each), and/or between the external reservoir 140 and the second reservoir. The device 110 operates substantially continuously, and has the added advantage of having few disruptions in mixtures sterility and providing a homogenous pegylated biological cells mixture.
In order to control the ratio of the first and second liquid mixtures micro-mixing within the mixing chamber 118, the cross-sectional area of the first tube 120 and the second tube 122 may be the same or different, as required. When the cross-sectional area of tubes 120 and 122 is the same, a substantially equal volume of the first and second liquid mixture is accordingly pumped into and mixed within the mixing chamber 118, assuming that the first and second peristaltic pumps 112, 113 are operating at a common rate. Given the same conditions, when the cross-sectional area of tubes 120 and 122 is different, a substantially unequal volume of the first and second liquid mixture is fed into and mixed within the mixing chamber 118.
When two peristaltic pumps 112, 113 are provided such as to independently control the output volume from the first and second reservoirs 114, 116 respectively, the volume ratio of the first and second liquid mixtures may also be different and varied, and thus controlled, by actuating the peristaltic pumps 112, 113 at different rotational speeds and thus at different output flow rates.
The mixing chamber 118 of the device 110 further comprises an outlet conduit 132 in fluid communication with both the mixing chamber 118 and a downstream a final reservoir 134, for evacuating the final solution mixture comprising polymer-modified biological cells from the mixing chamber 118.
Referring now to
Referring to
The presently described device enables the control and maintenance of the homogeneity of the ratio/mix of biological cells (such as red blood cells, for example) and the polymer (such as mPEG, for example) within a desired narrow band best suited for polymer grafting. As seen in
The polymer-modified cells described herein may include, but are not limited to, red blood cells, platelets, white blood cells, or any suspension of cells or cell aggregates such as pancreatic islets, and the like.
The embodiments of the invention described above are intended to be exemplary. Those skilled in the art will therefore appreciate that the forgoing description is illustrative only, and that various alternatives and modifications can be devised without departing from the spirit of the present invention. Accordingly, the present is intended to embrace all such alternatives, modifications and variances which fall within the scope of the appended claims.
