RESULTS AND DISCUSSION
Figure 1: (A) Determination of optimal seeding density on mesenchymal stem cell doubling time; (B) Determination of oxygen
levels on mesenchymal stem cell doubling time. (ALL FIGURES ARE COURTESY OF EMD MILLIPORE)
|
To better understand the growth of bone marrow-derived stem cells, initial experiments were designed to optimize growth rates
while the cells were grown two-dimensionally. Various seeding densities and oxygen levels were investigated to understand
which parameters led to the highest growth rate or lowest doubling time so that these seeding densities and oxygen levels
could later be used in the three dimensional (3D) culture (see Figure 1). To accomplish this, MSCs were grown over multiple
passages in tissue culture dishes at varying cell densities. A lower cell density of 5,000 cells per cm2 led to a shorter average doubling time of 49 h compared with cells seeded at a density of 20,000 cells/cm2, which resulted in a longer doubling time of 63 h. Differences in doubling times at different seeding densities could be
due to a number of factors, including inhibitory signals from neighboring cells, competition for growth factors and nutrients,
or waste product build up at higher density cultures. The seeding density of 5,000 cells per cm2 was selected as a seeding density for experiments designed to translate culture conditions from 2D to 3D culture.
To determine the best oxygen level to support MSC growth, cells were grown for multiple passages at different ambient oxygen
levels. Hypoxic conditions were preferable for cell growth, with a shorter average doubling time of 45 h for cells grown at
5% oxygen versus cells grown under normoxic conditions (i.e., 21% oxygen; doubling time of 54 h). The hypoxic conditions that
led to superior growth are likely closer to the physiological oxygen levels within bone marrow than are normoxic conditions
(3, 4). The depth of liquid in 2D culture is measured in millimeters, while 3D or deep cultures have vertical liquid levels
that can be measured in centimeters for spinner flasks and smaller stirred tank bioreactors like the Mobius CellReady 3L,
or meters for larger scale reactors. This height difference could cause lower dissolved oxygen levels in the spinner flask
than in 2D culture even when they are both exposed to normal atmospheric oxygen levels. Spinner flask cultures maintained
at different oxygen levels (5%, and 21%) showed similar growth (data not shown). For subsequent Mobius CellReady 3L cultures,
dissolved oxygen levels were monitored and shown to remain above 5% oxygen using headspace aeration. In addition to better
defining the desired operational window, these experiments led to the observation that a 48 hour doubling time was adequate
for 2D growth and this benchmark was used later to gauge 3D growth.
Figure 2: (A) Attachment and propagation of mesenchymal stem cells on various microcarriers; (B) Recovery of viable cells.
|
Different microcarrier types were evaluated for their ability to support MSC attachment, growth, and viable detachment (see
Figure 2). The attachment and propagation of MSCs on various microcarriers was first investigated. Four microcarriers showed
robust growth over the four day culture and were then further evaluated in the follow-up comparison. Top performers were compared
to reveal how efficiently viable cells could be harvested from the microcarriers. After five days of growth, cells were removed
from the microcarriers using three different dissociation reagents (e.g., trypsin, acutase, and collagenase) for 10 min at
37 °C and the results were averaged.
The collagen-coated microcarriers led to the highest viable cell recovery and were subsequently used in 3D studies. A high
percentage of viable cells were also recovered from Hilex microcarriers, and these microcrocarriers gave the best recovery
using an animal product-free microcarrier. The material of the microcarrier may have played a crucial role in promoting viable
recovery. Both the Hillex and collagen microcarriers are made of polystyrene, while the Cytodex beads are constructed from
dextran matrices. Even though the Cytodex beads showed a growth rate advantage over collagen beads, the number of viable cells
recovered was much higher from the collagen microcarrier culture. Because the stem cells are the product, the remainder of
the experiments were performed on the collagen-coated polystyrene microcarriers.
Following the attachment study, MSCs were cultured under stirred agitation conditions in spinner flasks. The MSCs were seeded
onto the collagen-coated microcarriers in ultra low adherent petri dishes under static conditions for two days, then transferred
to a spinner flask and agitated at thirty revolutions per minute. The MSCs were then sequentially passaged from one spinner
flask to another using 20% of the cells on microcarriers from the first spinner after five days and seeding them with 80%
of media with fresh microcarriers. This procedure was then repeated and a four- to six-fold increase in cell number was observed
over the first five days of the culture for each passage.
|
