important to separate the CDNs from the cell debris and other
interfering components to ensure the reliability and accuracy of
the downstream application [52]. Sephadex G-50 column will
separate the particles with different sizes and molecular weights
(size-exclusion) [53]; thus, this column is able to separate the
CDNs with the desired hydrodynamic size from other bigger
or smaller cellular debris or vesicles.
11. It is advisable to collect all fractions for protein quantifications
and physical characterizations for the first time of CDNs production. CDNs should possess relatively high protein amount
and appropriate hydrodynamic diameters.
12. The stability of CDNs were examined at 4 and 25
C. The size
of the exosomes was maintained around 150 nm at 4
C for up
to 7 days, whereas the size of CDNs increased drastically after
1 day stored at 25
C, indicating the instability due to possible
structural changes or degradation [29]. Our group has successfully performed the lyophilization of CDNs, as it is a commonly used technique for preservation and long-term stability
of biological samples, such as proteins and exosomes. It was
shown that lyophilized CDNs preserved physical characteristics
and functionality (unpublished results), which further corroborated the versatility of this CDNs production protocol.
13. The selection of dispersant is important for zeta potential
measurement, as the zeta potential measurement is affected
by the ionic strength and pH of the surrounding medium
[54, 55]. The rule of thumb for choosing the diluent for
the sample is to always perform a size measurement prior to
the zeta potential measurement. In this way, it is ensured that
the particles of interest are stable in the chosen buffer and no
aggregation or degradtion occur. For any physiological application, it is advisable to choose the buffer to maintain the pH at
the physiological range (pH 7.4) for a more accurate depiction
of the behavior of particles in physiological conditions. 0.1Â
PBS (10 mM PBS) is usually recommended for the routine
analysis. Thus, in this protocol, we performed a 10Â dilution
to our samples (in 1Â PBS) for the measurement.
14. The sample concentration may affect the size measurement. At
the low sample concentration, the light scattering may be
insufficient for the detection, while, at the high concentration,
multiple scattering may happen, leading to inaccuracy for the
size measurement [56]. It is advisable to keep the mean count
rate of the samples between 200 and 500 kilo counts per
second (kcps) to ensure an accurate measurement. For more
details on the setting of the measurement protocol, please refer
to the Malvern user manual. (https://www.malvernpanalytical.
com/en/learn/knowledge-center/user-manuals/
MAN0485EN
166
Yi-Hsuan Ou et al.
interfering components to ensure the reliability and accuracy of
the downstream application [52]. Sephadex G-50 column will
separate the particles with different sizes and molecular weights
(size-exclusion) [53]; thus, this column is able to separate the
CDNs with the desired hydrodynamic size from other bigger
or smaller cellular debris or vesicles.
11. It is advisable to collect all fractions for protein quantifications
and physical characterizations for the first time of CDNs production. CDNs should possess relatively high protein amount
and appropriate hydrodynamic diameters.
12. The stability of CDNs were examined at 4 and 25
C. The size
of the exosomes was maintained around 150 nm at 4
C for up
to 7 days, whereas the size of CDNs increased drastically after
1 day stored at 25
C, indicating the instability due to possible
structural changes or degradation [29]. Our group has successfully performed the lyophilization of CDNs, as it is a commonly used technique for preservation and long-term stability
of biological samples, such as proteins and exosomes. It was
shown that lyophilized CDNs preserved physical characteristics
and functionality (unpublished results), which further corroborated the versatility of this CDNs production protocol.
13. The selection of dispersant is important for zeta potential
measurement, as the zeta potential measurement is affected
by the ionic strength and pH of the surrounding medium
[54, 55]. The rule of thumb for choosing the diluent for
the sample is to always perform a size measurement prior to
the zeta potential measurement. In this way, it is ensured that
the particles of interest are stable in the chosen buffer and no
aggregation or degradtion occur. For any physiological application, it is advisable to choose the buffer to maintain the pH at
the physiological range (pH 7.4) for a more accurate depiction
of the behavior of particles in physiological conditions. 0.1Â
PBS (10 mM PBS) is usually recommended for the routine
analysis. Thus, in this protocol, we performed a 10Â dilution
to our samples (in 1Â PBS) for the measurement.
14. The sample concentration may affect the size measurement. At
the low sample concentration, the light scattering may be
insufficient for the detection, while, at the high concentration,
multiple scattering may happen, leading to inaccuracy for the
size measurement [56]. It is advisable to keep the mean count
rate of the samples between 200 and 500 kilo counts per
second (kcps) to ensure an accurate measurement. For more
details on the setting of the measurement protocol, please refer
to the Malvern user manual. (https://www.malvernpanalytical.
com/en/learn/knowledge-center/user-manuals/
MAN0485EN
166
Yi-Hsuan Ou et al.