Spheroids transfer

We and others have based invasion assays on preformed tumor spheroids generated either in hanging drops (88) or other nonadherent systems (61). These are then transferred to matrix monolayers or embedded in Matrigel and can be analyzed qualitatively or quantitatively, usually by microscopy and image analysis (see also Chapter 16). A96- or 1,536-well spheroid-based... 

Necessity of transferable (tight/robust) spheroids, no HT in case of nanoftbers... 

Fig. 1. Schematic overview of the tumor spheroid-based migration assay. Tumor spheroids (TS) are transferred from their cuiture vessei or piate into a 96-weii fiat-bottomed migration piate pre-coated with an extraceiiuiar matrix (ECM) protein of choice (in this case, geiatin). Digital images of the spheroids are then captured at t=0 and once every 24 h for a period of up to 72 h, exemplified here by CAL spheroids. Image analysis software is used to calculate the spheroid size and extent of migration. Scale bar=100 p.m.

Using a multichannel pipette (see Note 9), transfer one 4-day-old spheroid in a volume of 100 pL (see Note 10) into each well of the migration plate, resulting in a final volume of 300 pL/well (see Note 11). 

When the number of spheroids to analyze is relatively small (40-60 spheroids), the transfer of one spheroid per well can also be performed using a single channel rather than a multichannel pipette. If using disposable 200 pL pipette tips, it is advisable to cut off the ends using sterile scissors to increase the aperture and reduce the likelihood of disrupting the peripheral rim of cells on the spheroids. 

Depending on the method of spheroid generation, remove any excess culture medium to leave the spheroid in a volume of 100 pL ready for transfer to the migration plate. 

Naruse et al. proposed another bioreactor design [22,23], in which porcine hepatocyte spheroids are immobilized on non-woven polyester fabric. This device allows more direct contact between hepatocytes and perfused medium and improves, therefore, the mass transfer capacity. The non-woven fabric module expressed better metabohc and synthetic functions at 24 hours than a hollow fiber module containing spheroids in suspension culture. Longer term results are not yet available and the immunoexclusion properties of this fabric have not been addressed. 

Very few solutions have been obtained for heat or mass transfer to nonspherical solid particles in low Reynolds number flow. For Re = 0 the species continuity equation has been solved for a number of axisymmetric shapes, while for creeping flow only spheroids have been studied. 

Fig. 4.14 Factors to be used with Eqs. (4-61), (4-68), (4-69) and (4-70) for predicting heat and mass transfer to spheroids in creeping flow.

The mechanism of mass transfer to the external flow is essentially the same as for spheres in Chapter 5. Figure 6.8 shows numerically computed streamlines and concentration contours with Sc = 0.7 for axisymmetric flow past an oblate spheroid (E = 0.2) and a prolate spheroid (E = 5) at Re = 100. Local Sherwood numbers are shown for these conditions in Figs. 6.9 and 6.10. Figure 6.9 shows that the minimum transfer rate occurs aft of separation as for a sphere. Transfer rates are highest at the edge of the oblate ellipsoid and at the front stagnation point of the prolate ellipsoid. 

Fig. 6.11 Correlations and numerical calculations for heat transfer to spheroids and disks with Pr = 0.7.

No data are available for heat and mass transfer to or from disks or spheroids in free fall. When there is no secondary motion the correlations given above should apply to oblate spheroids and disks. For larger Re where secondary motion occurs, the equations given below for particles of arbitrary shape in free fall are recommended. 

Different eorrelations are required for three-dimensional bodies (spheres, disks, and spheroids) than for the two-dimensional shapes (cylinders and wedges). For three-dimensional shapes transfer in the aft region is correlated by... 

Heat a little sodium chloride in a platinum crucible to bright redness, and add a couple of drops of water to the hot crucible so that the water assumes the spheroidal state. In a moment, transfer the water to a beaker containing a faintly coloured soln. of blue litmus —the litmus is reddened, showing the presence of an acid—hydrochloric acid. The salt remaining in the crucible is dissolved in water, and it turns red litmus blue, showing the presence of an alkali—sodium hydroxide. There appears to be a reaction NaCl+H20 NaOH +HC1, and the water abstracts the more volatile hydrogen chloride. 

Encapsulation and Suspension. In this case, encapsulated spheroids of hepatocytes are contacted with blood in fluidized-bed, spouted-bed, and/or packed-bed systems. The mass transfer resistance should be high with encapsulated cells. However, the use of a suspension would lead to excessive shear forces being exerted on the cells. 

Transferring to spheroidal coordinates and employing the following identities, we have... 

In the physical literature, functional relationships for mass transfer can be found for a few cases of low Re, but unfortunately the actual range of dimensionless numbers for which the formulas are valid are often either not provided or can be somewhat misleading. For example, Clift et al. (1978) provide the following formula for a spheroid in creeping flow Re 1) ... 

Three main tendencies have been underlined in recent studies of structure and action mechanism ofbacterial photosynthetic reaction centers. The crystallographic structure of the reaction centers from Rps. viridis and Rb. spheroids was initially determined to be 2.8 and 3 A resolutions (Michel and Deisenhofer et al., 1985 Allen et al., 1986). Resolution and refinement of these structures have been subsequently extended to 2.2, 2.3 and 2.6 A. (Rees et al., 1989 Stowell et al., 1997, Fyfe and Johns, 2000 Rutherford and Faller, 2001). Investigations of the electronic structure of donor and acceptor centers in the ground and exited states by modern physical methods with a combination ofpico-and femtosecond kinetic techniques have become more precise and elaborate. Extensive experimental and theoretical investigations on the role of orbital overlap and protein dynamics in the processes of electron and proton transfer have been done. All the above-mentioned research directions are accompanied by extensive use of methods of sit-directed mutagenesis and substitution of native pigments for artificial compounds of different redox potential. 

The X-ray crystal structure of a reaction centre from Rhodobacter sphaeroides with a mutation of tyrosine M210 to tryptophan (YM210W) has been determined to have a resolution of 2.5 A (McAuley et al., 2000). It is shown that the main effect of the introduction of the bulkier tryptophan in place of the native tyrosine is a small tilt of the macrocycle of the (Bchl)L. The effect of the redox potential of the electron acceptor (Bchl) in RC from Rb. spheroides on the initial electron transfer rate and on the P (Bchl) population was investigated (Sporlein et al., 2000). Analysis of experimental... 

In the high-resolution ESR (326 GHz) study of the biradical state Qa - Qb - in the Rb. Spheroids, RC determines the exchange integral in the biradical (Jo = 109 s 1) (Calvo et al., 2001). Because the rate constant of electron transfer from Qa to Qb is essentially less (kET 104 s 1) (Feher et al., 1992 Xu et al, 2000) than expected for an nonadiabatic activationless ET and the kET values considerably deviate from the dependence of the supperexchange attenuation parameter (yET) on the distance between donor and acceptor centers in RCs (Fig. XXX), we can conclude that the ET is adiabatic and requires thermal activation. 

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