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Cell Attachment in vivo ( molecules involved) Cell attachment in vitro ( properties of substrata) Microcarriers
-Why microcarriers and desired property of microcarriers
Cell Attachment in vivo: Extra cellular Matrix proteins: Animal cells lack cell walls but are covered by an elaborate extracellular matrix (ECM) The ECM is made up of glycoproteins Collagen, proteoglycans, and fibronectin ECM proteins bind to receptor proteins in the plasma membrane called integrins
Functions of the ECM: i)Mechanical Support & Scaffolding, ii)Adhesion, iii)Movement, iv )Regulation of cell function
Schematic overview of the types of molecules that bind cells to each other and to the extracellular matrix( ECM)
Integrin Fibronectin
Integrin
Integrins Transduce Signals By Interacting With Many Signaling and Adaptor Proteins
Fibronectin
A large glycoprotein found in all vertebrates A dimer composed of two very large subunits joined by a pair of disulfide bonds near their carboxyl termini. Can bind to other ECM proteins AND to cells via different domains
3 Main Types of Signals 1. Soluble signals 2. Signals transmitted from cell to cell 3. Insoluble signals transmitted from ECM to cell Includes integrin-mediated adhesion and the responses to mechanical forces (shear, strain)
This involves divalent cations and glycoproteins (mainly fibronectin (Fn) and vitronectin) adsorbed to the culture surface. Under culture conditions the attachment glycoprotein originates from the serum supplement in the medium.
MHS (multivalent heparin sulphate) is synthesized by the cells. With anchorage-dependent cells proliferation occurs only after the spreading step.
Cell attachment, spreading and growth, suitable adhesive substrata is needed for anchorage depended cells
The common characteristics of suitable substrata are a) solidity, b) Charge density, c) wetting ability. a) Solidity : Cell will adhere to solid but not fluid phosphopholipid substrata and this due to a requirement for small, dense, rigid adhesive site in the substratum to match the focal contact of the cells. Fluid substrata would not allow the stable aggregation of such adhesive sites
b) Charge density : Charge density is expressed in terms of anion exchange capacity of substrata ( AEC). There are reports that 2.0 meq/g of dry DEAE shapadex particle is good cell attachment and growth. In Petri dishes 18meq/m2 is good for cell attachment and growth.
Collagens in the ECM present a very ordered array of charges and polar domains to the cells. Proteoglycans have also been shown to be arranged in regular pattern.
For good attachment and growth of cells , it requires a specific charge density pattern on polymer surface.
c) Wetting : Wetting is the ability of a drop of water to spread on a substratum indicated by the contact angle ( ) of the drop. In general, cells attach and spread better as the wetting ability of the substrata increases. Exceptions are agar and poly hydroxy ethyl methyl acrylate. Both wetting but completely non adhesive. Contact angle, (reflects the degree of wetting)
What are Microcarriers: Microcarrier Tiny spheres on which cells can attach and grow sphere allow adherent cells to be grown in suspension Greatly facilitates scale-up of adherent cell cultures by reducing the amount of space
needed
Designing Microcarriers: In the designing of microcarriers for cell attachment and growth, surface properties e.g. a)Chemical nature, b)charge density, c)roughness, d)wetability, e)rigidity are important factors.
Among these properties, the ion exchange capacity ( -NH2 content), hydrophobicity of polymer are considered a critical; factor for cell attachment and growth. Polymers used in microcarrier : *Aminated poly ( -methyl L-glutamate) [PG] *Cross linked poly(-lysine)[PL] particles *Poly allylamine [PAA] particles Cell attachment : Regarding cell attachment, adhesion of the extracelluar matrix would be one of the crucial parameter [ collagen, gelatin, proteoglycan, fibronectin etc.]. Interestingly,there is no correlation between the initial attachment rate constants and high cell growth observed.
Advantages of microcarrier cultures: This system has the following advantages over other methods of large-scale cultivation: High surface area to volume ratio can be achieved which, can be varied by changing the microcarrier concentration. This leads to high cell densities per unit volume with a potential for obtaining highly concentrated cell products. Cell propagation can be carried out in a single high productivity vessel instead of using many low productivity units, thus achieving a better utilization and a considerable saving of medium. Since the microcarrier culture is well mixed, it is easy to monitor and control different environmental conditions such as pH, pO2, pCO2 etc. Cell sampling is easy. Since the beads settle down easily, cell harvesting and downstream processing of products is easy. Microcarrier cultures can be relatively easily scaled up using conventional equipment like fermenters that have been suitably modified (Spier and Horaud 1985). Because of the many advantages of the technique itself, it has gained great popularity. Thus, a large variety of microcarriers are available in the market.
Types of Microcarriers
Surface
Microspheres
Macroporous
Cytodex
Based on dextran Bead formation Crosslinking by Epichlorhydrine, forming Sephadex G50 Sieving for narrow size distribution
Cytodex-1: Substitution of Sephadex G50 with positively charged N positively charged N-N-diethyl diethyl-amino amino- ethyl (DEAE) groups throughout the ethyl (DEAE) groups throughout the matrix
Cytodex-3 : Covalent coupling of gelatine (pig skin Covalent coupling of gelatine (pig skin type I collagen) onto the surface of type I collagen) onto the surface ofCytodex Cytodex 2
Bovine pulmonary artery cells grown on Bovine pulmonary artery cells grown on Cytodex Cytodex 3
Why Macroporous ?
Macroporous carriers allow serum, protein free perfusion (autocrine growth factors) shorter residence time of product at 37C support protection from stirrer, spin filter and air/O2 sparging allow cells to grow in 3D provide higher cell densities
Adherent cells 1ml macroporous carriers =1 Roller bottle (850 cm2)
Cytopore:Cotton cellulose (refined linter)- Cellulose solubilized cuprammoniumsulfate solution- Beads formed spray nozzle, cooled solution, -19C,macroporous beads formed Beads crosslinked with epichlorhydrine Cytopore 1 & 2 :Cytopore 1, 2 formed substitution macroporous particle DEAE (1.0, 1.85 meq/g) throughout matrix
live cells (flouresceinediacetate)
Cytoline: High Density Polyethylene (as base matrix), Silicate (increasing specific density, neg. charge ), NaCl crystals (determining pore size)
Pore size Length Thickness 10 - 400 m 1.5 - 2.5 mm 0.4 - 1.1 mm
Cytoline Cytoline 1
Sedimentation
velocity cm/min
Cytoline Cytoline 2
40-90
120-220
45-60
15-20
Density g/cm
1.32
1.03
Cytopilot concept
external circulation
internal circulation
After the reaction, the cross-linked particles were washed extensively with n-hexane, ethanol and pure water, and then filtered. All particles with a diameter of 100300 m were sieved out and used as microcarriers.
Cell attachment and growth of L929 cells onto neutral PG, animated PG, cross linked PL and cytodex 1 microcarrier
AEC(meq /g) pKa app
Anion exchange capacity [AEC] ( meq/g) : [poly ( -methyl L-glutamate)] PG-0.5 is 0.5; PG-3.3 is 3.3; poly(-lysine)PL-2.0 is 2.0, Pl-4.8 is 4.8 S6S0-1 - Cell number at day 6 divided by cell number at day 0
There is no correlation between the cell growth estimated at more than 2 days and the cell attached cell numbers estimated at 60 min, in all the above microcarriers..
2days
6days
2hr
2hr
PG-3.3
PG-3.3
PL-2.1
PL-2.1
Phase contrast micrographs of mouse L-929 cells on PG [poly ( -methyl L-glutamate)] -3.3 (A, C and G), PG-0.5 (D and H), PL [poly Lys]-2.1 (B, E and I) ,PL-4.8 (F and J) microcarriers.
2days
The micrographs AB, CF and GJ were captured at 2 h, 2 days and 6 days after inoculation, respectively. The only cells attached to the circumference of the microcarriers could be visualized because of the relatively low transparency of the polyamino acid based particles.
Original magnifications of the images were 400 (B, E and J) and 200 (others).
*Good correlation between cell growth and the pKa,app of the various particles was confirmed for both the PG and PL series. The PL-4.6 (pKa,app 7.0) showed high cell growth but the PL-1.3 (pKa,app 5.0) showed extremely low growth. The optimum pKa,app for cell growth in both particle series was near neutral pH.
Correlations between cell growth (S6S0-1 ) and the AEC (anion exchange capacity) (a) and pKa;app (b) on various microcarriers (PG, PL, PAA and Cytodex1). S6: total cell count attached to microcarriers after inoculation for 6 days; S0: inoculum
*Good correlation between cell growth and the pKa,app of the various particles was confirmed for both the PG and PL series. The PL-4.6 (pKa,app 7.0) showed high cell growth but the PL-1.3 (pKa,app 5.0) showed extremely low growth. The optimum pKa,app for cell growth in both particle series was near neutral pH. *Cytodex 1 with a DEAE moiety (pKa;app 7.5), showed relatively high cell growth. On the other hand, the PAA series with pKa;app ranging from 8.7 to 8.9, yielded no cell growth. These observations prove that cell growth is strongly dependent on the pKa;app but not necessarily on amino group content or the amino concentration on the microcarrier surface, which are in rough agreement with the AEC. *Cell growth is facilitated when the pKa;app value of the particles approaches neutral. The surface of particles with a neutral pKa was the most suitable environment for cell culture. On the other hand, the particles with high or low pKa values, evidenced cytotoxic properties.
Schematic illustration of the surface microenvironments on the aminated microcarriers for cell attachment and growth in terms of pKa,app: The pH microenvironment of surfaces is predominantly regulated by pKa;app due to buffer effects.
Initial attachment kinetics of L-929 cells onto the neutral PG based microcarriers (a) and cationic PG or PL based microcarriers (b).
A semilogarithmic plot of unattached cell concentration with respect to time yielded a straight line, indicating first-order attachment kinetics.
The order of the higher attachment rate constants was PG-C12, PL-2.0, PL-4.8bPG, PG-0.5>Cytodex1, PG- 3.3, PG-OH. The most hydrophobic particle with the longest alkyl chain, PG-C12, demonstrated the highest attachment rate constant (0.13 min1) exceeding that of other neutral particles, such as PG and PG-OH. However, hydrophilic particles with cationic charge, such as PL-2.0 and PL-4.8, also gave similarly high attachment rate constants. Among the aminated particles, the PL series showed higher attachment rate constants than the aminated PG series. These results suggest that a hydrophobic surface is advantageous in terms of cell attachment, on neutral polymer surfaces but not on charged surfaces.
* No correlation between the initial attachment rate constants and high cell growth was observed. The particles that facilitated cell growth, PLA4.8 and PG-0.5, gave high and low attachment rate constants, as did the lethal particles, PG-3.3 and PL-2.0. RTICLE
The hydrophobicity or flexibility of side chains seemed to be advantageous for cell culture, on neutral surfaces.
Cell growth on cationic particles having primary amino groups was drastically dependent upon the AEC.
The surface of particles with a neutral pKa,app was the most suitable environment for cell growth, because of the neutral pH microenvironment. In other words, the particles with low and high pKa values possessed acidic and basic pH surface microenvironments,which had cytotoxic effects, respectively. No correlation between attachment rate constants and high cell growth was observed.
Ref : The design of polymer microcarrier surfaces for enhanced cell growth.Biomaterials 24 (2003) 42534264