System for in-situ management of pH of culture

Abstract

Disclosed is a hydrogel composition loaded with a base that regulates and maintains the pH of cell culture media. The invention particularly discloses in situ pH managing hydrogels (pHmH) loaded with base Mg(OH).sub.2 for pH control in small scale cultures comprising adherent mammalian cells, suspended mammalian cells, microorganisms, a microorganism with plasmids and the like thereof.

Claims

1. A system for in situ management of pH of a culture comprising a carrier and a base, wherein said base is Mg(OH).sub.2 or Ca(OH).sub.2, and said carrier is selected from the group consisting of a hydrogel, a polymer matrix comprising a silicone elastomer, an encapsulation for said base, a membrane and a coating film, wherein release rate of said base is dependent on pH of the culture, said carrier is non-responsive to pH change of said culture, and wherein the carrier is loaded with the base.

2. The system of claim 1, wherein the carrier is a hydrogel.

3. The system of claim 2, wherein the hydrogel comprises 2-hydroxyethyl methacrylate (HEMA) as monomer; Azoisobutyronitrile, (AIBN) as initiator and optionally ethylene glycol dimethacrylate (EGDMA) as cross linker.

4. The system of claim 3, wherein an amount of the HEMA is from 71.8 to 99.9% by weight of total amount of the hydrogel.

5. The system of claim 4, wherein the base is loaded in the carrier in a manner selected from the group consisting of: the base is dispersed in the carrier, and the base is included in a reservoir within the carrier.

6. The system of claim 1, wherein said culture comprises medium and cells selected from the group consisting of unmodified animal cells, genetically modified animal cells, microbial cells, insect cells, stem cells and mesenchymal stem cells.

7. The system of claim 1, wherein said culture is grown in a medium comprising glucose, and optionally a buffer.

8. The system of claim 1, wherein the pH of the culture is managed between 5-9.

9. The system of claim 1, wherein the pH of the culture is managed for a period of up to 15 days.

10. The system of claim 1, wherein Mg(OH).sub.2 or Ca(OH).sub.2 is released depending on pH of the culture.

11. The system of claim 1, wherein Mg(OH).sub.2 or Ca(OH).sub.2 is released depending on pH of the culture, and wherein said pH dependent-release of Mg(OH).sub.2 or Ca(OH).sub.2 is independent of the carrier.

12. The system of claim 1, wherein the in situ management does not require periodic measurement of pH.

13. The system of claim 1, wherein the system comprises two discs of the carrier.

14. A process for in situ management of pH of a culture, the process comprising employing a system comprising; a carrier selected from the group consisting of a hydrogel, a polymer matrix comprising a silicone elastomer, an encapsulation for said base, a membrane and a coating film, wherein said carrier is non-responsive to pH change of said culture; and a base, wherein said base is Mg(OH).sub.2 or Ca(OH).sub.2, wherein release rate of said base is dependent on pH of the culture, and the carrier is loaded with the base.

Description

DESCRIPTION OF FIGURES

(1) FIG. 1 depicts Effect of pH on release of Mg.sup.+2 from pHmH discs. (a) One pHmH-1 was added to 100 mL of 200 IBM HEPES solution at pH 6 (cross), 7 (square), or 8 (circle), respectively. Samples were taken every day to measure Magnesium concentration. N=3. Error bars indicate one standard deviation above and below the average. (b) Release rate of Mg.sup.+2 from pHmH-1 at pH 6, 7, and 8. FIG. 1c depicts pHmH-3 of a different composition in comparison to pHmH-1 showing release rate can be changed by changing parameters like cross linking density, geometry of the hydrogel.

(2) FIG. 2 depicts percent swelling of pHmH-1 at different pH,

(3) FIG. 3 depicts effect of pHmH-1 disc addition on pH profile during culture of suspension CHO cells. CHO cells were seeded in shake flasks in 20 mL CD CHO medium containing 8 mM glutamine without antibiotics at 0.310.sup.6 cells/mL and fed with glucose and protein hydrolysate at times indicated by solid arrows. Four grams per liter hydrolysate was added at the first feed, and 2 g/L at subsequent feed additions. pHmH-1 disc was added to the culture at time(s) indicated by dotted arrows. Number of pHmH-1 discs added is indicated next to the dotted mow. Cultures were run in duplicates. (a) Addition of single pHmH-1 disc. (b) Staggered addition of two pHmH-1 discs. (c) Control (without pHmH-1 disc). Error bars indicate one standard deviation above and below the average,

(4) FIG. 4 depicts effect of pHmH-1 disc addition on pH of adherent A549 culture. Cells were seeded in 8 mL DMEM:F12 containing 10% FBS in 25 cm.sup.2 TC flasks and incubated at 37 C., 5% CO.sub.2, with two pHmH-1 discs (square) or without (circle) pHmH-1 disc. Cultures were sampled to measure pH, glucose and lactate concentrations. N=3, SD.

(5) FIG. 5 depicts (a) Growth of E. coli K12 in LB-glucose with and without pHmH-2 (b) Glucose consumption of E. coli K12 in LB-glucose with and without pHmH-2 (c) pH profiles of E. coli K12 cultures in LB-glucose with and without pHmH-2. Control, without pHmH-2 .diamond-solid., With single pHmH-2 .square-solid. and with staggered addition of two pHmH-2 .circle-solid.,

(6) FIG. 6 depicts (a) Growth of E. coli K12 in Modified Wilms and Reuss medium (no buffer) with and without pHmH-2 (b) Glucose consumption of E. coli K12 in Modified Wilms and Reuss medium (no buffer) with and without pHmH-2 (c) pH profiles of E. coli K12 cultures in Modified Wilms and Reuss medium (no buffer) with and without pHmH-2. Control, without pHmH-2 .diamond-solid., With single pHmH-2 .square-solid. and with staggered addition of two pHmH-2 .circle-solid.,

(7) FIG. 7 depicts (a) Growth of E. coli K12 in Modified Wilms and Reuss medium with 50 mM MOPS, with and without pHmH-2 (b) Glucose consumption of E. coli K12 with and without pHmH-2 (c) pH profiles of E. coli K12 with and without pHmH-2. Control, without pHmH-2 .diamond-solid., With single pHmH-2 .square-solid. and with staggered addition of two pHmH-2 .circle-solid., simultaneous addition of two pHmH-2

(8) FIG. 8 depicts (a) Growth of E. coli K12 in Modified Wilms and Reuss medium with 200 mM MOPS, with and without pHmH-2 (b) Glucose consumption of E. coli K12 with and without pHmH-2 (c) pH profiles of E. coli K12 with and without pHmH-2. Control, without pHmH-2 .diamond-solid., With single pHmH-2 .square-solid. and with staggered addition of two pHmH-2 .circle-solid., simultaneous addition of two pHmH-2 .

(9) FIG. 9 depicts (a) Growth of TOP10 E. coli cells transformed with a 5 Kb plasmid, in LB-Glucose with and without pHmH-2 addition. (b) Glucose consumption (c) pH profiles. Without poly-HEMA pHmH-2 .diamond-solid., With staggered addition of two pHmH-2 .square-solid. (d) Plasmid DNA yields from cultures with and without pHmH-2 loaded with Mg(OH).sub.2,

ABBREVIATIONS

(10) pHmH: pH managing hydrogel

(11) HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

(12) HEMA: 2-hydroxyethyl methacrylate

(13) EGDMA: Ethylene glycol dimethacrylate

(14) AIBN: Azoisobutyronitrile

(15) DMEM: F12 medium: Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12

(16) FBS: Fetal bovine serum

(17) CD: Chemically Defined

(18) CHO: Chinese Hamster Ovary

(19) A549 cells: human lung carcinoma

(20) MOPS buffer: 3-(N-morpholino) propanesulfonic acid buffer

DETAILED DESCRIPTION OF INVENTION

(21) The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

(22) In accordance with the objectives of the invention, the inventors provide a system comprising a carrier and a base for in-situ management of pH.

(23) In a preferred embodiment, the invention provides a hydrogel as carrier, wherein the carrier is loaded with base which manages/maintains pH of cell culture media without the need of any additional infrastructure.

(24) The additional infrastructure includes monitoring pH or measuring pH through, mathematical model, robotic instruments, Optical Transmittance, dye indicator and like thereof.

(25) In preferred embodiment, the invention provides base loaded hydrogel composition for in-situ regulation and maintenance of the pH in small scale culture formats without pH measurements, comprising a monomer, an initiator, a base and optionally a cross linker. According to the embodiment above the monomer is 2-hydroxyethyl methacrylate (HEMA); the cross-linker is ethylene glycoldimethacrylate, (EGDMA); the initiator is Azoisobutyronitrile, (AIBN) and the base is Mg(OH).sub.2.

(26) In a preferred embodiment, the base is Mg(OH).sub.2.

(27) The inventors have selected magnesium hydroxide as a base for pH maintenance due to its low solubility product (Ksp=1.810.sup.11), that might reduce leakage of base which is important for long term culture, and for its effect on osmolarity, since the release of two OH.sup. is accompanied by only one Mg.sup.+2. Also, as for any other base, the solubility of magnesium hydroxide increases with a decrease in pH, which is expected to result in an increase in hydroxyl ion release rate with decreasing pH.

(28) Further in the instant hydrogel composition, the base is dispersed in the hydrogel, or the base is within the hydrogel as a reservoir. Further the base is included as a layer between two layers of the hydrogel. It may be further evident that the release of the base from the carrier is dependent on the pH of the culture.

(29) In an embodiment, the culture is grown in a medium comprising glucose.

(30) In an optional embodiment, the culture is grown in a medium comprising buffer. With reference to FIGS. 5, 6, demonstrating cultures with no buffer and FIGS. 7 and 8 demonstrating cultures with buffer, the improved growth of respective cells is demonstrated with pHmH-2.

(31) In another preferred embodiment, the invention provides method for preparation of base loaded hydrogels comprising steps of;

(32) a) mixing of monomer 2-hydroxyethyl methacrylate (HEMA) and optionally a cross-linker, ethylene glycol dimethacrylate, EGDMA, in suitable molar ratio;

(33) b) dissolving an initiator Azoisobutyronitrile, (AIBN) to solution in step a);

(34) c) sterilizing the obtained solution from step b) by filtering through a 0.22 m filter;

(35) d) adding sterilized base i.e. Mg(OH).sub.2 to solution in step c); and

(36) e) allowing to polymerize the mixture in step d) in a at temperature in the range of (60 to 90) for 2-3 hrs under aseptic condition to obtain pH managing hydrogel (pHmH) disc. In an embodiment, the pHmH may be prepared and then sterilized by means well known in the art such as autoclaving, gamma irradiation and such like.

(37) According to the invention, the monomer 2-hydroxyethyl methacrylate (HEMA) and a cross-linker, ethylene glycol dimethacrylate, (EGDMA) are mixed, wherein the molar ratio of (HEMA:EGDMA) is present in the range of 80:20 to 100:0; preferably 85:15 to 93:7 mol/mol of (HEMA:EGDMA) in the presence of an initiator i.e. Azoisobutyronitrile, (AIBN) in the concentration ranging from 0.1% to 0.8%; preferably 0.5%. Further the solution containing HEMA, EGDMA with dissolved AIBN is sterilized by filtering through a 0.22 nm filter, subsequently the sterilized Mg(OH).sub.2, is added to the above solution and the mixture is allowed to polymerize in a glass disc at temperature in the range of 60 to 90 C. for 2-3 hours under aseptic conditions to obtain pH managing hydrogels (pHmH) discs, which are washed in water for 2-4 days to remove unreacted/unpolymerized monomer.

(38) In another embodiment, the instant pHmH discs are prepared preferably in molar ratio of 93:7 mol/mol of HEMA:EGDMA.

(39) In yet another embodiment, the invention provides in-situ pH managing hydrogel (pHmH) loaded with Mg(OH).sub.2 that maintains pH of cell culture at least between 0.3 units of desired pH, without the need for measurement of pH. The prepared hydrogel in the form of disc used for in situ pH maintenance for cell culture in small scale formats, wherein the cell culture is selected from the group consisting of mammalian cells in suspension, insect cells, adherent mammalian cells, microbial cells, microbial cells expressing plasmids.

(40) The small-scale culture formats are not limited to tissue-culture flasks, shake flasks or spinner flasks.

(41) The mammalian cell in suspension is selected from the group of unmodified or genetically modified Chinese Hamster Ovary (CHO) cell line, human bone marrow-derived mesenchymal stem cells, (hBMMSCs), mouse hybridoma cell line, mouse myeloma cell line, Baby Hamster Kidney (BHK) cell line, human embryonic kidney (HEK) cells, HEK 293, PER.C6 and human retinal cells,

(42) The adherent mammalian cell is selected from the group of unmodified or genetically modified A549 cells, CHO, BHK, HEK 293, PER.C6, mouse myeloma cells, stem cells and primary cells derived from human tissue.

(43) The microbial cell is selected from the group consisting of unmodified or genetically modified Pseudomonas, Salmonella, Geobacter, Bacillus, Pichia, Saccharomyces, Hansenuia and Escherichia coli cells.

(44) In an another embodiment, the mammalian cells in suspension is particularly culture of a suspension Chinese Hamster Ovary (CHO) cell line in CD CHO medium, whereas the adherent mammalian cells are A549 cells in DMEM:F12 containing 10% FBS, the microbial cell is E. coli K12 cells and the microbial cells expressing plasmids are E. coli TOP10 cultures transformed with plasmid DNA.

(45) In yet another embodiment, the pHmH of the invention maintains the pH of medium within 0.3 units of the set point. The pH of suspension cells is maintained at 0.2 units of desired pH; wherein the desired pH is in the range of 7 to 7.5.

(46) Accordingly (pHmH) hydrogel maintain the pH in between 6.8 and 7.2 for suspension Chinese Hamster Ovary (CHO) cell line in CD CHO medium.

(47) Further the instant pH managing hydrogels (pHmH) loaded with magnesium hydroxide were evaluated in a fed batch culture of a suspension Chinese Hamster Ovary (CHO) cell line to assess the culture pH profile as compared to control cultures, where pH was allowed to drift, as is typical in shake flasks. pHmH disc addition allowed pH to be maintained between 6.8 to 7.2 for suspension CHO cells in CD CHO medium, the in situ pH control using the pHmH described herein, can increase the utility of shake flasks as a screening tool for animal cell based bioprocesses without the need for any additional infra structure.

(48) In yet another embodiment, the pHmH of the invention maintains the pH of adherent cells at 0.1 units of desired pH; wherein the desired pH is in the range of 7 to 7.5.

(49) The instant hydrogel maintain the pH between 7.3 and 7.5 for adherent A549 cells in DMEM:F12 containing 10% FBS in tissue culture flasks.

(50) In another embodiment of the invention, the pHmH is used to increase biomass yields for microbial culture as exemplified in herein below. As embodied herein and with reference to the examples provided, the pHmH manages and maintains the pH of the medium without the need for periodic requirement of measurement of the pH of the medium. The pH is maintained within the desirable range for a period of up to 15 days.

(51) In an another embodiment, the base loaded hydrogel maintains the pH of microbial cell culture comprising of bacterial cells particularly Escherichia coli.

(52) Accordingly the invention provides magnesium hydroxide loaded hydrogels for pH management to maintain pH in permissive range for optimal growth of E. coli K12 cell cultures. E. coli cells produce acetic acid under conditions of excess glucose due to overflow metabolism resulting in a decrease in culture pH. In the instant invention, the inventors demonstrate the use of magnesium hydroxide loaded hydrogels to increase the biomass yield for E. coli K12 and volumetric plasmid yield in an E. coli TOP10 strain transformed with a plasmid.

(53) 2 Hydroxy ethyl methacrylate (HEMA) (97% pure), Ethylene glycol dimethylacrylate (EGDMA) and Dichlorodimethylsilane were purchased from Sigma Aldrich (MO, USA) and Azoisobutyronitrile, the thermal initiator was purchased from Spectrochem (India). E. coli K12 and a clone of TOP10 E. coli (Invitrogen Corporation, CA, USA) transformed with a 5 kb plasmid cells were used. GOD/POD glucose assay kit was purchased from Beacon diagnostics, India. QIAprep Spin Miniprep Kit from QIAGEN (Hilden, Germany) was used for plasmid DNA purification.

(54) Media Composition and Culture Conditions:

(55) LB broth consists of 10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl. 20 g/L glucose was added to LB broth after autoclaving to serve as carbon source. For the culture of TOP10 E. coli cells, culture medium was supplemented with 100 g/ml ampicillin.

(56) The modified Wilkins and Reuss medium was prepared as described by (Scheidle et (al., 2011) and consists of 20 g/L glucose; 5 g/L (NH.sub.4).sub.2SO.sub.4; 0.5 g/L NH.sub.4Cl; 3 g/L K.sub.2HPO.sub.4; 2 g/L Na.sub.2SO.sub.4; 0.5 g/L MgSO.sub.4.7H.sub.2O; 0.01 g/L thiamine hydrochloride; 1 mL/L trace element solution (0.54 g/L ZnSO.sub.4.7H.sub.2O; 0.48 g/L CuSO.sub.4.5H.sub.2O; 0.3 g/L MnSO.sub.4.H.sub.2O; 0.54 g/L CoCl.sub.2.6H.sub.2O; 41.76 g/L FeCl.sub.3.6H.sub.2O; 1.98 g/L CaCl.sub.2.2H.sub.2O; 33.39 g/L Na.sub.2EDTA (Titriplex III). 3-(N-morpholino)propanesulfonic acid (MOPS) was added at indicated concentrations in some experiments.

(57) Analytical Methods:

(58) Growth was monitored by measuring the optical density (550 nm) at regular intervals after appropriate dilution. pH of the culture supernatant was measured using a pH electrode (Sentek, UK). Samples were stored at 20 C. and glucose concentration was measured using GOD/POD glucose assay kit (Beacon Diagnostics, India). All experiments were performed in triplicates.

(59) Swelling Kinetics:

(60) The empty pHmH was dried in an oven at 50 C. for a day to complete dryness to study the swelling kinetics. The weight of pHmH was measured before and after immersing in water to determine the extent of swelling. The swollen weight was measured at several instances after removing the surface water with blotting paper. Swelling ratio was calculated using the formula:
Swelling ratio=(W.sub.sW.sub.d)/W.sub.d,
where W.sub.d is the weight of the dry pHmH and W.sub.s is the weight of the swollen pHmH.

(61) A pHmH xerogel was added to water, and the increase in weight of the pHmH was measured as a function of time to estimate swelling kinetics. Empty pHmH reached equilibrium swelling in approximately 4 hours. The pHmH reached an equilibrium swelling ratio of 67%.

(62) The present invention is illustrated herein below with examples, which are illustrative only and should not be construed to limit the scope of the present invention in any manner.

EXAMPLES

Example 1

Preparation of Hydrogel (pHmH-1)

(63) Poly-HEMA hydrogels were synthesized using monomer, 2-hydroxyethyl methacrylate [HEMA, 97% pure] and a cross-linker, ethylene glycol dimethacrylate, EGDMA (Sigma-Aldrich Corp, USA) at 93:7 mol/mol HEMA: EGDMA in the presence of an initiator Azoisobutyronitrile, AIBN (0.5%, Sigma Aldrich Corp, USA). The solution containing HEMA, EGDMA with dissolved AIBN was sterilized by filtering through a 0.22 m filter. 100 mg sterile Mg(OH).sub.2, sterilized by autoclaving at 15 psi and 121 C. for 20 minutes, was added to 500 l of the above solution and the mixture allowed to polymerize in a 1.3 cm diameter glass disc at 75 C. for 3 hours followed by 90 C. for 15 minutes under aseptic conditions. These discs, referred to hereafter as pH managing hydrogels-1 (pHmH-1), were washed in water for three days

Example 2

Preparation of Hydrogel (pHmH-2)

(64) 0.5% w/v Azobisisobutyronitrile (AIBN) was dissolved in 1.5 ml 97% pure 2-Hydroxyethyl methacrylate. After dissolution, the mixture was poured onto a glass surface. 150 mg of a Mg(OH).sub.2 was added to the solution and mixed. The gel mixture was allowed to polymerize at 75 C. for 3 hours. The hydrogels prepared had a surface area of 23 cm.sup.2. The hydrogels were washed in water for one day to remove any unreacted monomer. These discs are referred to hereafter as pH managing hydrogels-2 (pHmH-2)

Example 3

Preparation of Hydrogel (pHmH-3)

(65) 0.5% w/v Azobisisobutyronitrile (AIBN) was dissolved in 1.5 ml 97% pure 2-Hydroxyethyl methacrylate. After dissolution, the mixture was poured onto a glass surface. 100 mg of a Mg(OH).sub.2 was added to the solution and mixed. The gel mixture was allowed to polymerize at 75 C. for 3 hours. The hydrogels prepared had a surface area of 23 cm.sup.2. The hydrogels were washed in water for one day to remove any unreacted monomer. These discs are referred to hereafter as pH managing hydrogels-3 (pHmH-3)

Example 4

Rate of Release of Mg+2 from pHmH at Different pH

(66) The release of Magnesium from the pHmH-1 of the invention over different pH and the management of the pH and its maintenance in culture medium is evaluated, wherein the release rates of OH.sup. can be changed by changing parameters like cross linking density, geometry of the hydrogel and the identity of the base, with reference to FIG. 1, it is observed that release of Mg.sup.+2 from pHmH discs is dependent on pH even though the hydrogel is not pH sensitive (Refer FIG. 2).

(67) One pHmH-1 was added to 100 mL of 200 mM HEPES solution at pH 6, 7 or 8 respectively, and incubated under shaking conditions at 37 C. Experiment at each pH was performed in triplicates. Samples were taken once every day for the measurement of Mg.sup.+2 concentrations as described below. The pH of the solution was measured everyday to ensure no change. For the experiment at pH 6, pH was adjusted on days 8 by addition of H.sub.2SO.sub.4.

(68) Mg+2 Release from pHmH at pH 6, 7 and 8

(69) FIG. 1 shows the cumulative Mg.sup.+2 released from the pHmH-1 at different pH values. The release rate of [OH.sup.] is calculated from this data based on stoichiometry. The total amount of Mg.sup.+2 released over typical culture duration is thus a small fraction of the initial loading of magnesium hydroxide in the pHmH-1. The slope of the graph of Mg+2 released vs. time in FIG. 1a provide estimates of the release rate of Mg+2 at the different pH. The release rate can be seen increasing with decreasing pH (FIG. 1b). Since HEMA:EGDMA hydrogels are not known to be pH responsive, this pH responsive behavior of the release rate is probably observed due to the phenomenon of increasing solubility of a base with decreasing pH.

(70) pHmH-3 hydrogels were added to phosphate buffers at different pH to evaluate release of magnesium at different pH values.

(71) Phosphate buffer comprised NaH.sub.2PO.sub.4 and Na.sub.2HPO.sub.4 in the following amounts, dissolved in 500 ml water: for pH 8-1.4 g and 24.97 g respectively, pH 7-7.836 g and 15.466 g respectively, pH 6-12.143 g and 3.217 g respectively pH 5-13.614 g and 0.45 g

(72) Gels were pre-swollen in water and added to the 500 ml phosphate buffers at the different pH, and maintained at 37 C. with shaking at 200 rpm. All experiments were run in triplicate. Samples were withdrawn at regular intervals. Mg concentration was measured using SPECTRO ARCOS SOP Inductively Coupled Plasma spectrometer (SPECTRO Analytical Instruments, Germany). With reference to FIG. 1c, the release rate of Mg.sup.+2 increases with decrease in pH.

(73) The FIG. 1a and is clearly shows that the release rate of the base may be tailored by varying the composition of the carrier.

Example 5

Evaluation of pH Maintenance on Suspension CHO Cell Culture

(74) A suspension CHO cell line (Inbiopro) was used to evaluate the ability of the pHmH-1 discs as exemplified in example 1 to maintain pH in suspension cells during a fed batch culture. Cells were inoculated at a density of 0.310.sup.6 cells/ml in 20 ml CD CHO medium (Invitrogen. Corporation, CA, USA) containing 8 mM glutamine, without antibiotics in shake flasks (37 C., 10% CO2, 110 rpm) and fed with glucose and protein hydrolysate at indicated times. 4 g/l hydrolysate was added at the first feed, and 2 g/l at subsequent feed additions. Glucose concentrations are shown in FIG. 3. Hydrogels were added on two schedules (a) Single pHmH-1 added on day 3 (b) Staggered addition: The first pHmH-1 disc was added on day 3, the second pHmH-1 disc was added at the time of the second feed. Samples were taken every day for measuring cell density, viability, glucose and lactate concentrations (the cells make lactate which affects pH) and pH. Cell density was quantified using a haemocytometer. Viability was assessed using a tryptan blue dye exclusion assay. Glucose and lactate concentrations were measured on a YSI Biochemistry analyzer. 100-150 l of the cell supernatant after centrifugation of the sample was allowed to reequilibrate with 10% CO.sub.2 in the incubator. pH was immediately measured using a pH probe (Sentek, UK).Control cultures showed decrease in pH to 6.4 towards the end of culture. Staggered addition of two pHmH discs enabled pH to be largely maintained between 6.8 to 7.2 while culture viability was greater than 85% (FIG. 3).

Example 6

Evaluation of pH Maintenance on Adherent Cells

(75) pHmH-1 discs were also evaluated for pH maintenance for adherent cells using A549 as a model system.

(76) A549 cells were seeded in 8 ml DMEM:F12 medium with 2 g/l sodium bicarbonate and 10% FBS without antibiotics in 25 cm.sup.2 tissue culture flasks with two pHmH-1 discs. Control cultures were likewise seeded in triplicate without the addition of pHmH-1 disc. All flasks were incubated at 37 C. under 5% CO.sub.2. 150 l samples were withdrawn everyday for measuring pH. Cells were observed under an inverted phase contrast microscope to visually confirm cell growth.

(77) The pH in the control flasks decreased over seven days (FIG. 4), while that in the flasks with two pHmH-1 discs was maintained between 7.3 to 7.5 over seven days (FIG. 4).

Example 7

Preparation of Poly-HEMA Hydrogels Loaded with Glucose

(78) Gels made were the polymers of 0.5%. Azobisisobutyronitrile (AIBNas initiator) solution (w/v) in Hydroxyethylmethacrylate (HEMA) with 5% v/v Ethylene glycol dimethylacrylate (EGDMA as cross linker). Inner surfaces of glass tubes (with flat bottom) of 1.4 mm inner diameter were coated with Silanization solution (5% dichlorodimethylsilane (v/v) in heptane), to make the surface hydrophobic. These were then dried in oven for 15 minutes at 50 C. These were then filled with 625 mg glucose and 50 mg Mg(OH).sub.2. 600 l of HEMA+EGDMA+AIBM mixture was added to 625 mg glucose+50 mg Mg(OH).sub.2 and allowed to polymerize in water bath for 1 hour at 75 C. The gels formed were then removed off the tube by breaking the tube, washed for half an hour in RO water. Before addition to media, they were surface sterilised with 70% ethanol, rinsed well in sterile water and used.

Example 8

pH Management in E. coli K12

(79) To check the effect of addition of pHmH-2 in E. coli K12 cultures, hydrogel was added to the culture medium at indicated times. E. coli K12 was cultured in 20 ml Modified Willms and Reuss medium containing 200 mM MOPS and glucose as the carbon source. The modified Willms and Reuss medium was prepared as described by (Scheidle et al., 2011) consists of 20 g/L glucose; 5 g/L (NH.sub.4).sub.2SO.sub.4; 0.5 g/L NH.sub.4Cl; 3 g/L K.sub.2HPO.sub.4; 2 g/L Na.sub.2SO.sub.4; 0.5 g/L MgSO.sub.4.7H.sub.2O; 0.01 g/L thiamine hydrochloride; 1 mL/L trace element solution (0.54 g/L ZnSO.sub.4.7H.sub.2O; 0.48 g/L CuSO.sub.4.5H.sub.2O; 0.3 g/L MnSO.sub.4H.sub.2O; 0.54 g/L CoCl.sub.2.6H.sub.2O; 41.76 g/L FeCl.sub.3.6H.sub.2O; 1.98 g/L CaCl.sub.2.2H.sub.2O; 33.39 g/L Na.sub.2EDTA. 3-(N-morpholino) propanesulfonic acid (MOPS) was added at indicated concentrations in some experiments.

(80) The pHmH-2 hydrogels used in the study were autoclaved at 15 psi and 121 C. for 20 minutes before their addition in E. coli K12 cultures. The cultures were maintained in a shaker incubator at 37 C. with a shaking speed of 250 rpm. Hydrogel was not added to control cultures. Hydrogels were added at (a) Single pHmH-2 at beginning of culture, (b) One at the beginning and one 4 hours after inoculation and (c) Two pHmH-2 at the beginning of the culture. All the culture flasks were sampled at regular intervals for the measurement of optical density at 600 nm.

(81) Cultures grown in Modified Wilms and Reuss medium containing 200 mM MOPS reached an optical density of 5.2 (FIG. 8). A single pHmH-2 was added at the time of inoculation in all except the control culture. Two pHmH-2 were added at the time of inoculation to culture with simultaneous addition of two hydrogels. Staggered addition of hydrogels, one hydrogel at the time of inoculation and another at about 4 hours from time of inoculation allows the optical density to reach a maximum value of 14 with a threefold increase in final biomass yield.

(82) With reference to FIG. 6, the fore mentioned experiment was conducted with no MOPS, FIG. 7 is with 50 mM MOPS. It may be concluded that while the hydrogels promote better cell growth in all cases, addition of buffer further improves pH management of culture. Also, it may be concluded that staggered addition of pHmH as described herein may be resorted to for better culture performance and retains the pH management efficiency of the system.

Example 9

pH Management in E. coli for Plasmid Production

(83) The effect of addition of pHmH-2 on plasmid yield in an E. coli strain transformed with a plasmid was investigated. Luria Bertani broth consists of 10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl. 20 g/L glucose was added to LB broth to serve as carbon source.

(84) This strain was grown in 20 ml LB medium containing 20 g/l glucose and 100 g/ml ampicillin. To compare the plasmid yield cultures of E. coli were grown with and without pHmH-2. One pHmH-2 was added at the beginning of the culture and another one was added 2 hours after inoculation. After 8 hours of inoculation, 1 ml of sample was taken from control flasks and from the flasks containing hydrogels. These samples were harvested by centrifugation at 10,000 rpm for 5 min, the medium was decanted and the pellet was stored at 80 C. for further processing for plasmid DNA purification. Plasmid DNA was purified using QIAprep Spin Miniprep Kit from QIAGEN, Hilden, Germany. FIG. 9 shows the plasmid yields from control cultures and pHmH-2 added cultures. The purified Plasmid DNA was quantified by measuring the absorbance at 260 nm using a NanoDrop 2000 Spectrophotometer.

(85) With reference to FIG. 5, LB medium with 20 g/lt glucose was inoculated with E. coli K 12.

Example 10

Addition of pHmH-3 and Glucose Loaded Hydrogel

(86) E. coli TOP 10 transformed with plasmid was cultured in LB with addition of both glucose loaded hydrogels and pHmH-3. Control cultures were cultured in LB with no hydrogels added and harvested after 14 hours.

(87) 50 ml cultures of E. coli TOP10 were cultured in LB supplemented with 100 g/ml ampicillin in 250 ml shake flasks. Cultures were inoculated at an OD of 0.1 and were incubated at 250 rpm at 37 C. When the culture OD reached approximately 2, only the glucose loaded hydrogel or the glucose loaded hydrogels and pHmH-3 were added respectively. Cultures were incubated for a further 15 hours. Control cultures with no hydrogel addition were inoculated from an overnight culture in LB+ ampicillin by a 500-fold dilution and cultured further for 14 hours. 1 ml of sample was taken at harvest.

(88) Table 1 lists the final OD.sub.550, pH and plasmid yields in the three conditions. The cultures with both the glucose loaded hydrogels and pHmH-3 showed a 348% increase in OD and a 394% increase in plasmid yields compared to the control cultures. The pH at the end of the culture was similar to the control.

(89) TABLE-US-00001 TABLE 1 Effect of hydrogel addition on volumetric plasmid yield. OD.sub.550, final culture pH and volumetric plasmid yield in control cultures in LB, and cultures in LB with glucose loaded hydrogels with pHmH-1. 95% confidence intervals are reported. Plasmid yield OD.sub.550 Final pH (g/ml) control 2.5 0.1 8.6 0.1 7.7 1.9 With glucose 11.2 0.6 8.2 0.2 38.0 2.4 loaded gel and pHmH-3 With glucose 3.8 0.2 5.1 0.04 5.9 0.7 loaded hydrogel

ADVANTAGES OF INVENTION

(90) 1. In-situ management of pH 2. Enables increase in biomass and/or product 3. Applicable to adherent mammalian cells, mammalian cells in suspension, microbial cells, microbial cells expressing plasmids