Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
2001-02-21
2002-04-30
Prats, Francisco (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S042000, C435S173100, C435S173200, C435S173400, C435S173500, C435S173600, C435S174000, C435S176000, C435S177000, C435S180000, C435S242000, C435S383000, C435S383000
Reexamination Certificate
active
06379956
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention concerns a method for populating substrates with living biological cells and populating devices that can be used therefor.
2. Background Description
For various purposes, it is desired to populate substrates or matrices with cells which should then grow on those substrates. Both substrates of natural origin and those of synthetic origin are considered. Examples of substrates of natural origin might, for instance, be collagen substrates. Synthetic substrates could, for instance, be biologically inert plastics. The substrates to be populated with living cells often have a net-like or sponge-like structure to make it easier for the cells to grow into them.
For populating, the cells must be taken to the substrate without being damaged or destroyed; and they must be applied to the substrate in such a manner that growth can occur. In most cases, one tries for a closed cell lawn.
The cultured cells, or cells removed elsewhere, are generally in a culture medium. In the usual inoculation processes, usually done on the laboratory scale, the substrate is brought into contact with the cells in the culture medium, for instance, in a “roll flask”. The roll flask is rolled in a rolling incubator, so that the substrate, the culture medium, and the cells are maintained in constant but not overly vigorous motion.
With inoculation there is the problem that the substrate must be flushed by the culture medium with the cells, because cells should be transported continuously to the surface, but on the other hand, the flow, or the rolling or pivoting motion within the flask must not be so great that the freshly applied cells are swept off again before they can successfully become established. There is also the problem that the substrate should be accessible to the cells at all points. Therefore it must not fold because of the rolling motion, or stick to the roll flask.
In previous inoculation experiments, there was little or no ability to attain normal differentiation of the growing cells. The artificially inoculated substrates are therefore distinct from the natural precursors in the structure of their cell layers.
Another disadvantage of previous inoculation methods was that complete confluence of the endothelial cell lawn could not be developed in bioartificial vessels.
It would be desirable, then, for a process to be developed in which the cells used for inoculation were enabled by the culture conditions to develop cell colonies typical of the tissue, and to develop physiological differentiation.
SUMMARY OF THE INVENTION
The invention is, therefore, based on the objective of developing an inoculation method and inoculating means usable for it, with which a substrate can be inoculated under optimal growth conditions. The inoculating means to be developed should provide effective inoculation without expensive apparatus. Furthermore, handling of the substrate should be reliable and simple during the entire inoculation process.
The inoculating means should preferably be usable simultaneously as a vessel for sterilization, acellularization [see Note 2], storage, transportation, and frozen preservation. That is desired to avoid breaking the chain of sterility up to the end user.
This objective is attained by a process for inoculating substrates with biological cells
in which the substrate held within a device and the cells provided for inoculation are subjected to at least one inoculation phase, during which the substrate is rotated about at least one axis in or with the device and the cells are repeatedly brought into contact with the substrate during the rotary movement,
and in which the substrate is subjected to at least one perfusion phase during or after one or more inoculation phases, during which the substrate is perfused in at least regions of its external or internal surface.
By inoculation phase we mean, with respect to this invention, a phase in cells with which the substrate is to be inoculated are taken to the substrate in a suitable manner.
Preferably, during the inoculation phase, cells are moved to the substrate in a medium or by streaking on the substrate, insertion into the substrate, or coating of the substrate with a medium containing the cells (e. g., collagen). In this process the cells can be provided with factors or cofactor, especially growth factors or chemotactic factors.
By inoculation phase we mean, with respect to this invention, a phase during which the substrate is perfused, as in the usual expert terminological use of the concept “perfusion”. Preferably, the substrate is perfused with a liquid nutrient medium for cell culture, blood, or plasma, which can in turn be enriched with various substances.
Multiple inoculation and perfusion phases can take turns with each other during the process, preferably alternately. While carrying out the inoculation phase in a similar manner was already known, it was found, surprisingly, that incorporation of one or more perfusion phases in the process distinctly improves the result of the inoculation. Use of the perfusion phases simulates quasiphysiological conditions which are important for development of normal cell differentiation. Shear stress produced to the extent intended by the perfusion, differentiates endothelial cells. Smooth muscle cells react to pulsatile pressures, which can also be generated during the perfusion. Oxygen and nutrients can be provided during the perfusion, and pressure stresses can be applied. Physiological pressure fluctuations, such as occur in vivo between systole and diastole are particularly important for orientation of the newly formed cells (of the extracellular matrix) and development of normal stability and ability to bear pressure stress. Inoculation of a synthetic tubular vessel on its interior has already been described in Br. J. Surg. 1991, Vol. 78, 878-882. The inoculation is done with aliquots of an endothelial cell suspension while the vessel is rotated. In that work it was determined that the inoculation of the PTFE [poly(tetrafluoroethylene); Teflon] vessel coated with fibronectin could be significantly improved in its ability to withstand pulsing flowing blood if, instead of a short (20-minute) inoculation cycle, a longer cycle was used overnight in a rolling incubator. However, it was not recognized there that intentional perfusion of the inoculated vessel can itself improve the result of inoculation. Furthermore, the device described there can be rotated only about its own axis and not in many spatial directions. Also, mounting and dismounting of the vessel are intricate, as caps must be removed from both ends. In removal of the finished inoculated vessel there is a danger that the structure will become unsterile because several intricate manipulations are required to remove the vessel from the reactor.
It is preferable for the rotation of the substrate during the inoculation phase to be done in a superimposed rotary motion about at least two spatial axes. This can also be a randomly controlled rotation in all spatial directions, which will be considered further in relation to suitable inoculating devices for the process. Alternatively, the rotation of the substrate during the inoculation phases can be about at least two spatial axes successively or alternately.
The process can also be set up so that the substrate is subjected to a supplementary resting phase after at least one inoculation phase. Such a resting phase can be inserted to provide the partially or completely inoculated substrate a stress-free consolidation phase during which the cell lawn can strengthen itself or even reorganize internally.
The intermittent perfusion also allows development of multilayer structures. That can be done, for instance, in multiple inoculation phases, adding first connective tissue cells, smooth muscle cells, and then endothelial cells, with perfusion phases between the individual inoculation phases. Thus the following cell types can be applied to already structured associations differentiated typically for th
Srivastava Kailash C.
Whitham, Curtis & Christofferson
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