Microcarriers for cell culture
OwnerEmma Petiot, Carlos Chocarro +1inventors
InventorsEmma Petiot, Carlos Chocarro, Edwin-Joffrey Courtial
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Abstract
Methods for producing microcarriers by ink jetting, comprising propelling a droplet of a solution comprising one or more biomaterials capable of forming a hydrogel and one or more cells, into a liquid medium. Microcarriers produced by such methods, methods in which such microcarriers are used to culture a cell population in a bioreactor and a system for producing microcarriers by ink jetting are also disclosed.
Claims15
A method of producing a microcarrier for cell culture, the method comprising:
- providing a solution comprising one or more biomaterials capable of forming a hydrogel and one or more cells;
- and
- propelling a droplet of the solution into a liquid medium by ink jetting, thereby producing a cellularised microcarrier.
The method of any preceding claim, wherein the solution further comprises a liquid culture medium and/or wherein the solution has been obtained by mixing one or more solutions each comprising biomaterials capable of forming a hydrogel and/or cells in a liquid culture medium, and/or wherein the solution further comprises a compound or composition that promotes cell adhesion, optionally wherein the compound or composition is selected from fibrinogen, Dermial®, hyaluronic acid, chitosan, collagen I, and combinations thereof.
The method of any preceding claim, wherein the droplet has a diameter between 100 μm and 2 mm, between 100 μm and 1.5 mm, between 100 μm and 1 mm, optionally between 100 μm and 800 μm, and/or wherein the droplets comprise a volume of solution below 5 μl, below 1.7 μl, below 0.5 μl, or between 0.5 nl and 0.3 μl.
The method of any preceding claim, wherein the method further comprises consolidating the droplet, optionally wherein consolidating the droplet comprises exposing the droplet to one or more solutions that cross-link one or more of the biomaterials capable of forming a hydrogel and/or wherein consolidating produces a microstructure comprising a hydrogel matrix and the one or more cells, and/or wherein the liquid medium in which the droplet is propelled comprises one or more compounds capable of cross-linking one or more of the biomaterials capable of forming a hydrogel.
The method of any one of the preceding claims, wherein the step of propelling a droplet of the composition is performed:
- (i) at a temperature between 2° and 40° C., or between 3° and 40° C.;
- (ii) using an inkjet printer with a fluid pressure between 0.25 and 5 bar, between 2 and 5 bar, optionally between 3 and 4 bar;
- and/or
- (iii) using an ink jetting device with a valve opening time between 5 and 50 ms, optionally about 10 ms, and/or wherein the droplet is propelled using a flow rate between about 9 μL/s and about 20 μL/s, or between 15 μL/s and 20 μL/s.
The method of any preceding claim, wherein the biomaterial capable of forming a hydrogel comprises alginate and/or gelatin, optionally wherein the solution comprises at most 5% w/v gelatin or at most 3% w/v of gelatin, and/or wherein the solution comprises alginate and gelatin, optionally wherein the solution comprises between 2% and 10% w/v of gelatin, and between 0.5% and 3% w/v of alginate, and/or wherein the solution comprises about 5% w/v gelatin and about 2% w/v alginate, about 2.5% w/v gelatin and about 2% w/v alginate.
The method of any preceding claim, wherein the viscosity of the solution comprising one or more biomaterials capable of forming a hydrogel and one or more cells is below 5 Pa·s, below 1 Pa·s or between 0.05 and 0.7 Pa·s.
The method of any preceding claim, wherein the method comprises collecting the droplet in a non-static liquid medium, optionally wherein the liquid medium is in an agitated vessel such as a bioreactor, and/or wherein the liquid medium into which the droplet is propelled is agitated, optionally with an impeller.
The method of any preceding claim, wherein:
- the solution comprises about 2% w/v fibrinogen, about 2.5% w/v alginate, and about 2.5% w/v gelatin, and wherein the step of propelling a droplet of the composition is performed at a temperature between 30° C. and 40° C. and/or in conditions such that the viscosity of the solution is between 0.06-0.12 Pa·s;
- or
- the solution comprises about 2% w/v fibrinogen, about 2% w/v alginate, and about 5% w/v gelatin, and wherein the step of propelling a droplet of the composition is performed at a temperature between 30° C. and 40° C. and/or in conditions such that the viscosity of the solution is between 0.15 and 0.63 Pa·s;
- or
- the solution comprises about 2% w/v fibrinogen, about 1% w/v alginate, and about 10% w/v gelatin, and wherein the step of propelling a droplet of the composition is performed at a temperature between 35° C. and 40° C. and/or in conditions such that the viscosity of the solution is between 0.11-0.63 Pa·s.
The method of any preceding claim, wherein propelling a droplet of the composition is performed using a continuous ink jetting device, and/or wherein propelling a droplet of the composition is performed using a device comprising a piezoelectric valve, and/or wherein propelling a droplet of the composition is performed in a sterile environment, and/or wherein propelling a droplet of the composition is performed using an apparatus configured such that every element of the apparatus flow path is sterilisable.
The method of any preceding claim, wherein propelling a droplet comprises applying pressure on the solution passing through a nozzle, optionally wherein the nozzle has a diameter between about 50 μm and about 300 μm, between about 50 μm and about 200 μm, or between about 50 μm and about 100 μm and/or wherein the nozzle has a diameter of about 50 μm and the droplet has a diameter between 100 μm and 400 μm, or wherein the nozzle has a diameter of about 100 μm and the droplet has a diameter between 400 μm and 1000 μm.
A cellularised microcarrier obtained or obtainable through the method of any preceding claim.
A method for performing a bioproduction process comprising the production of a cell population, wherein the process comprises maintaining a microcarrier according to claim 18 in suspension in a culture medium in a culture vessel, optionally wherein the culture vessel is an agitated bioreactor and/or wherein the method further comprises culturing the cells for at least 3, 5, 7, 14, 21, or 28 days, or about 14, days, 21 days, 28 days, 35 days, 42 days, 49 days, 60 days, about 1 month or about 2 months, and/or maintaining the cells for at least 4, at least 10 or at least 20 amplifications, and/or wherein maintaining the cells comprises maintaining the cells at least partially embedded in the microcarrier in suspension in the culture medium without adding additional physical support for cell growth, and/or wherein the cells are stem cells and wherein culturing the cells comprises maintaining the cells in an undifferentiated state.
A system comprising:
- a bioreactor, optionally wherein the bioreactor is a stirred tank;
- an inkjet printing device coupled with the bioreactor or with a microcarrier recovery tank fluidically connected with the bioreactor, wherein the inkjet printing device, bioreactor and optionally microcarrier recovery system are all connected in a single sterile system and wherein the inkjet printing device is configured to dispense droplets of liquid directly into a liquid medium in the bioreactor or in the microcarrier recovery tank.
Dependencies are inferred from the claim text.