U.S. patent application number 11/955744 was filed with the patent office on 2008-05-01 for plate and method for high throughput screening.
This patent application is currently assigned to Biolex Therapeutics, Inc.. Invention is credited to KEITH EVERETT.
Application Number | 20080102518 11/955744 |
Document ID | / |
Family ID | 23133387 |
Filed Date | 2008-05-01 |
United States Patent
Application |
20080102518 |
Kind Code |
A1 |
EVERETT; KEITH |
May 1, 2008 |
PLATE AND METHOD FOR HIGH THROUGHPUT SCREENING
Abstract
A multiple well plate and method for media exchange, including a
body defining a plurality of cell wells each connected via a
channel to one of a plurality of aspiration holes, is provided. The
cell wells contain a porous, hydrophilic frit which is suspended on
a ledge above a reservoir of fluid media and supports a tissue
sample. The properties of the frit wick the fluid media upwards to
supply the tissue sample with nutrients for growth and
proliferation. Old media is aspirated from the wells by a liquid
handling device which inserts a pipette tip into the aspiration
holes. The pipette tip applies a suction pressure which draws the
media out of the cell well, through the channel, into the
aspiration hole and out through the pipette tip. New media is
dispersed through the pipette tip and directly into the cell
well.
Inventors: |
EVERETT; KEITH; (Raleigh,
NC) |
Correspondence
Address: |
ALSTON & BIRD LLP
BANK OF AMERICA PLAZA
101 SOUTH TRYON STREET, SUITE 4000
CHARLOTTE
NC
28280-4000
US
|
Assignee: |
Biolex Therapeutics, Inc.
|
Family ID: |
23133387 |
Appl. No.: |
11/955744 |
Filed: |
December 13, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10157562 |
May 29, 2002 |
7326385 |
|
|
11955744 |
Dec 13, 2007 |
|
|
|
60294430 |
May 30, 2001 |
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Current U.S.
Class: |
435/288.4 ;
702/19 |
Current CPC
Class: |
C12M 33/06 20130101;
B01L 2300/0829 20130101; B01L 3/50255 20130101; B01L 2400/049
20130101; B01L 3/5085 20130101; Y10T 29/49996 20150115; Y10T
436/2575 20150115; B01L 2200/026 20130101; B01L 3/5025 20130101;
B01L 2200/12 20130101; C12M 25/04 20130101; B01L 2200/0642
20130101 |
Class at
Publication: |
435/288.4 ;
702/019 |
International
Class: |
C12M 1/00 20060101
C12M001/00; G01N 33/48 20060101 G01N033/48 |
Claims
1. A method of using a multiple well plate having a body with an
upper surface, the body defining a plurality of first holes and a
plurality of second holes, each hole having an upper edge defined
by the upper surface and each of the first holes connected in fluid
communication with a respective one of the second holes, said
method comprising: placing a frit into each of the plurality of
first holes; placing a tissue into each of the plurality of first
holes and onto the frit; dispensing a media into each of the
plurality of first holes; inserting a pipette into each of the
plurality of second holes; and aspirating the media from the first
holes by applying a suction pressure to the second holes using the
pipette, the suction pressure drawing the media from the first
hole, into the second hole and into the pipette so as to flush the
media from the multiple well plate.
2. A method of using the multiple well plate of claim 1, further
comprising re-dispensing the media into each of the plurality of
first holes after aspirating the media from the first holes.
3. A method of using the multiple well plate of claim 1, further
comprising removing a lid from the multiple well plate before
placing the frit and replacing the lid after aspirating the
media.
4. A method of using the multiple well plate of claim 1, wherein
dispensing a media into each of the plurality of first holes
includes dispensing the media into the respective ones of the
second holes using the pipette.
5. A method of using the multiple well plate of claim 1, wherein
said body further defines a channel connecting each of the first
holes to the respective one of the second holes in fluid
communication and wherein said aspirating draws the media through
the channel.
6. A method of using the multiple well plate of claim 1, wherein
the tissue includes a plant tissue.
7. A method of using the multiple well plate of claim 1, wherein
the tissue is a duckweed tissue.
8. A method of using a frit material to support a tissue for
growth, comprising: suspending the frit material above a supply of
nutrients; wicking the nutrients through a porous, hydrophilic
structure of the frit material; and supplying the nutrients through
a top surface of the frit to the tissue so that the tissue is
continuously supplied with nutrients and growth of the tissue is
promoted.
9. A method of claim 8, further comprising storing up to 550 .mu.l
of nutrients in the frit before supplying the nutrients.
10. A method of exchanging a fluid media in a multiple-well plate
defining a plurality of wells each connected in fluid communication
with an adjacent one of a plurality of aspiration holes, the method
comprising: positioning a plurality of dispensing pipette tips each
over a respective one of the plurality of wells of the
multiple-well plate; dispensing media out of each of the dispensing
pipette tips and into the respective well over which the dispensing
pipette tip is positioned; repeating positioning and dispensing out
of the dispensing pipette tips until all of the wells of the
multiple-well plate are filled; inserting a plurality of aspirating
pipette tips each into a respective one of the plurality of
aspiration holes; aspirating media using each of the aspirating
pipette tips to suction the media out of the respective aspiration
hole in which the aspiration tip is positioned; washing the
aspirating pipette tips at a washing station; and repeating
inserting, aspirating and washing until all of the wells of the
multiple well plate are emptied of the media.
11. A computer program product for controlling exchange of fluid
media in a multiple-well plate defining a plurality of wells each
connected in fluid communication with an adjacent one of a
plurality of aspiration holes, the computer program product
comprising a computer-readable storage medium having
computer-readable program code portions stored therein, the
computer-readable program code portions comprising: a first
executable code portion for positioning a plurality of dispensing
pipette tips each over a respective one of the plurality of wells
of the multiple-well plate; a second executable code portion for
dispensing media out of each of the dispensing pipette tips and
into the respective well over which the dispensing pipette tip is
positioned; a third executable code portion for repeating
positioning and dispensing out of the dispensing pipette tips until
all of the wells of the multiple-well plate are filled; a fourth
executable code portion for inserting a plurality of aspirating
pipette tips each into a respective one of the plurality of
aspiration holes; a fifth executable code portion for aspirating
media using each of the aspirating pipette tips to suction the
media out of the respective aspiration hole in which the aspiration
tip is positioned; a sixth executable code portion for washing the
aspirating pipette tips at a washing station; and a seventh
executable code portion for repeating inserting, aspirating and
washing until all of the wells of the multiple well plate are
emptied of the media.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. application
Ser. No. 10/157,562, filed May 29, 2002, which claims the benefit
of U.S. Provisional Application No. 60/294,430, filed May 30, 2001,
both of which are hereby incorporated herein in their entirety by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to devices and methods for
promoting the growth of tissue in experimental and production
settings, more particularly the use of specialized plates to house
the tissue and the cycling of media to biologically sustain the
tissue.
BACKGROUND OF THE INVENTION
[0003] High-throughput screening typically requires parallel
processing of batches of samples, typically in multiple well plates
(MWPs) of 24, 48, 96, and 384, or more, wells per plate. MWPs are
standard sizes that can be used with existing high-throughput
machinery, such as with robotic-controlled pipetters. Each
pipetting station of a robotic controlled pipetter employs
pipetting heads having an array of pipette tips that address
multiple wells simultaneously. Although used effectively for the
screening of liquid samples, the current multiple well plates are
generally ineffective for screening plant and other tissues, and
the secretory products associated with these tissues, that require,
or prefer, more complex environments such as solid support
structures.
[0004] For example, attempts have been made to grow plants in MWPs
by suspending the plants in a liquid media within each well.
However, the plant tissue is deprived of oxygen when sitting in the
liquid, effectively "drowning" the plant tissue in an anaerobic
environment. Other attempts have been made using media that are
generally more solid and provide a substrate on which the plant
tissue may be supported above the fluid, such as a gel or filter
paper disk. Although these types of supports avoid drowning the
plants, they are difficult to exchange and replenish when the
nutrients or media have been depleted. Paper bridges doused in
liquid media have also been used as tissue supports and the liquid
media is somewhat more easily replenished. However, empirical
evidence has shown that paper bridges are difficult to manage in an
automated system and are generally ineffective at promoting plant
tissue growth. Without being wed to any particular theory, this may
be because the liquid media does not easily penetrate the paper
bridge (i.e., the paper bridge is only mildly hydrophilic) and the
tissue supported thereon lacks a continuous supply of media.
[0005] A common approach to supplying fresh media to plant tissue
is to move the plant tissue to a container holding fresh media.
Movement of the plant tissue is a relatively slow and labor
intensive process, as multiple plates must be replenished and
otherwise prepared for each batch of plant tissue. In addition
there is a threat of loss or contamination of the tissue samples
when they are removed from the wells.
[0006] Another approach to aspirating and removing spent media and
tissue byproducts is to use an assay plate having a plurality of
wells, with each well having a hole or port at the base of the
well. A filter is positioned at the bottom of each well to support
the tissue. Spent media can be vacuum harvested from each well
through the port using a vacuum manifold assembly. One example of a
vacuum manifold assembly is the MultiScreen Vacuum Manifold system
manufactured by MILLIPORE of Bedford, Mass. The assay plate rests
on a manifold that supplies a vacuum which draws the media through
the filter disks, out of the cell wells and through the ports,
where it is captured in the manifold below. Although the filter
disks in the assay plate allow media to be drawn out of the plate,
it is difficult for the filter disks to retain enough media to
support tissue maintenance and growth for any length of time.
Because the ports at the bottom of the wells are open to the
ambient air, the ports may allow media to leak or evaporate and may
also provide a path for microbial contamination of the wells. In
addition, the wells of the assay plate cannot be individually
sampled because the vacuum manifold harvests the media from all of
the wells at once.
[0007] The tissue of animals, and other types of organisms may also
require, or prefer, solid support structures that inhibit the use
of multiple well plates and high-throughput screening techniques.
For instance, the growth of cartilage cells may be promoted by the
use of a collagen fibril matrix that simulates an in vivo
environment. Similar to the plant tissue discussed above, the
cartilage cells need a supply of fresh media that is replenished at
various intervals to survive and/or proliferate. In addition, some
of the cartilage cells proliferate within the collagen fibril
matrix and cannot be moved independent of the matrix. Moving the
cells to a new plate with a fresh supply of media requires movement
of the entire collagen matrix which is a relatively slow and
inefficient process that exposes the tissue to contamination.
[0008] It would be advantageous to have a multiple well plate that
allows the use of high throughput screening methods for non-liquid
samples. In addition, it would be advantageous to have a multiple
well plate that allows the use of high throughput screening methods
for tissues that require, or prefer, solid support structures. It
would be further advantageous to have a multiple well plate that
promotes the growth of tissue, such as plant tissue, without posing
the risk of drowning the tissue in liquid media or allowing the
tissue to dehydrate or become contaminated. It would also be
advantageous to have a multiple well plate that allows media to be
easily replenished, without undue disturbance of the tissue
contained in the wells. Additionally, it would be advantageous to
have the capability of sampling less than the total number of wells
in the plate without disturbing the unsampled wells.
SUMMARY OF THE INVENTION
[0009] The present invention addresses the above needs and achieves
other advantages by providing a multiple well plate (MWP) and
method for media exchange that promotes the growth of plant tissue,
and other types of tissue, by controlling the supply of media to
the tissue and allowing for the regular exchange (removal and
addition) of media without disturbing the tissue. The MWP includes
an array of wells, with each well being coupled with an adjacent
aspiration hole that allows media to be aspirated from the wells
using a conventional, automated pipette head. The MWP and pipette
head provide a virtually complete exchange of the spent media
because of the novel dual-well architecture. A hydrophilic, porous
frit housed within each well supports the tissue and holds the
media in its interstices, allowing contact between the tissue and
the media while avoiding an anaerobic condition. The media is
wicked upwards in sufficient quantities to provide nutrients to the
tissue and promote proliferation of the tissue.
[0010] In one embodiment, the invention includes a plate for
holding a porous frit that supports a tissue. The porous frit is
saturated in a media that may be regularly aspirated and refreshed,
for example, by a top-loading pipette device. The plate comprises a
body with an upper surface defining a first hole and a second hole.
The first hole has a first hole upper edge defined by the upper
surface of the body and a first hole bottom portion defined within
the body and below the upper surface of the body. The first hole is
configured to receive the porous frit and the tissue, and to hold
the media bathing the porous frit and the tissue. The second hole
has a second hole upper edge defined by the upper surface of the
body and a second hole bottom portion defined within the body and
below the upper surface of the body. The second hole bottom portion
is in fluid communication with the first hole bottom portion so
that the pipette device can access the second hole upper edge to
aspirate the media by applying a vacuum. The pipette device also
refreshes the media by adding fresh media directly onto the frit in
each well.
[0011] In another aspect, the body of the plate further defines a
passage connecting the first hole bottom portion and the second
hole bottom portion in fluid communication. The body may further
include a ledge protruding into the first hole bottom portion for
supporting the frit above a reservoir of fluid. The body may also
define a plurality of the first and second holes, with each first
hole in fluid communication with a respective one of the second
holes to form a MWP. In another aspect, the body defines an array
of first and second holes, for example 12, 24, 48, 96, 384, or 1536
first and second holes, wherein each first hole is in fluid
communication with a respective one of the second holes. The first
and second holes preferably have cylindrical shapes.
[0012] In yet another aspect, the top surface of the body is
configured to receive a cover plate disposed thereon. Preferably
the cover plate is transparent to light transmission and the first
hole upper edge is configured to also allow light transmission,
thereby promoting plant tissue growth.
[0013] In another embodiment, the present invention includes a
method of making a plate for holding a porous frit that supports a
tissue wherein the porous frit is saturated in a media. The media
is regularly aspirated and refreshed by a pipette device to promote
proliferation of the tissue. The method includes providing a body
with an upper surface and defining a first and second holes in the
body. Defining a first hole in the body includes drilling through
the upper surface of the body to form a first hole upper edge and
drilling below the upper surface to form a first hole bottom
portion. The second hole is defined by drilling through the upper
surface of the body to form a second hole upper edge and drilling
below the upper surface to form a second hole bottom portion. The
first and second hole bottom portions are connected in fluid
communication by forming a passage in the body and between the
first hole bottom portion and the second hole bottom portion.
Preferably, the passage is formed by inserting a saw disc into the
first hole bottom portion and moving the saw laterally until
encountering the second hole bottom portion.
[0014] In yet another embodiment, the present invention includes a
method of using a MWP. A frit is placed into each of the plurality
of first holes and tissue, preferably a duckweed or other plant
tissue, is placed onto the frit. A media is dispensed into each of
the plurality of first holes. A pipette is inserted into each of
the plurality of second holes and used to aspirate the media from
the first holes. The media is aspirated by applying a suction
pressure to the second holes using the pipette. The suction
pressure draws the media from the first hole, into the second hole
and into the pipette so as to flush the media from the plate. Fresh
media can be re-dispensed into the plurality of first holes after
aspirating the media from the second holes.
[0015] In still another embodiment, the present invention includes
a frit material for supporting a tissue having a porous structure,
a top surface and a bottom surface. The porous structure has
hydrophilic properties and a plurality of interstices. The top
surface is configured to support the tissue. The bottom surface is
in fluid communication with a reservoir of fluid media. The
hydrophilic properties of the porous structure wick the fluid into
its interstices so that the supported tissue is supplied with
sufficient liquid media from the reservoir to promote growth of the
tissue.
[0016] The present invention has several advantages. For example,
the tissue samples in the wells do not have to be moved or
disturbed when the provided media is spent, cutting down on
workload and ensuring sterile and optimal growth conditions. The
plates may be used with conventional liquid handling pipette heads
of the fixed tip or individually controllable tip versions because
the aspiration holes are accessible from the upper surface of the
body, i.e., a top-loading arrangement. The use of robotic liquid
handlers with the plate promotes a well-to-well consistency in the
treatment of the tissue, as well as the efficient removal and
replacement of the media. The top-loading aspect allows the use of
a standard lid for sterility control and removes the need for a
separate vacuum manifold station for pulling out media. The lack of
a manifold allows for the differential treatment of each well and
provides flexibility in liquid handler design and selection, as
well as experimental model and sample interrogation functions. The
liquid head can be configured to remove the media as well as add
new media with no change of tooling or pipette tips.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Having thus described the invention in general terms,
reference will now be made to the accompanying drawings, which are
not necessarily drawn to scale, and wherein:
[0018] FIG. 1 is a plan view of a MWP for liquid media exchange of
a first embodiment of the present invention;
[0019] FIG. 2 is an enlarged, cross-sectional view of a single well
and an aspiration hole from the MWP of FIG. 1;
[0020] FIG. 3 is a perspective view of a tip seal of another
embodiment of the present invention;
[0021] FIG. 4 is a perspective view of a system for replenishing
media in the MWP shown in FIG. 1 of another embodiment of the
present invention;
[0022] FIG. 5 is a flow chart of a method of replenishing media
using the system of FIG. 4; and
[0023] FIG. 6 is a sectional view of the well and aspiration hole
of FIG. 2 being machined in another embodiment of the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present invention now will be described more fully
hereinafter with reference to the accompanying drawings, in which
preferred embodiments of the invention are shown. This invention
may, however, be embodied in many different forms and should not be
construed as limited to the embodiments set forth herein; rather,
these embodiments are provided so that this disclosure will be
thorough and complete, and will fully convey the scope of the
invention to those skilled in the art. Like numbers refer to like
elements throughout.
[0025] A multiple well plate (MWP) 10 of the present invention
includes a body 12 having an upper surface 14. The body 12 defines
an array of first holes, or wells 16 and an array of second
aspiration holes 22, as shown in FIG. 1. A plurality of channels 28
each connect a respective one of the wells 16 to an adjacent one of
the aspiration holes 22, as shown in FIG. 2. In one embodiment,
disposed in each of the wells 16 is a porous, hydrophilic frit 30
which supports a tissue sample 32 over a reservoir of liquid media
36. The porous, hydrophilic properties of the frit 30 wick the
media 36 upwards, so as to supply the media to the tissue sample
32. Exchange of old, depleted media 36 is facilitated by the
aspiration holes 22 which are each sized and configured to receive
a 10 pipette tip 34. During aspiration, several of the pipette tips
are inserted into the aspiration holes 22 and apply a vacuum
pressure. The vacuum pressure draws the fluid media 36 out of each
of the wells 16, through the channels 28, through the adjacent one
of the aspiration holes 22 and into the pipette tips.
[0026] The tissue sample 32 is preferably a plant tissue, such as
dicot and monocot tissue such as tissue from corn (Zea mays),
Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly
those Brassica species useful as sources of seed oil, alfalfa
(Medicago sativa), rice (Oryza sativa), rye (Secale cereale),
sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl
millet (Pennisetum glaucum), proso millet (Panicum miliaceum),
foxtail millet (Setaria italica), finger millet (Eleusine
coracana)), sunflower (Helianthus annuus), safflower (Carthamus
tinctorius), wheat (Triticum aestivum), soybean (Glycine max),
tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts
(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium
hirsutum), sweet potato (Ipomoea batatus), cassaya (Manihot
esculenta), coffee (Coffea spp.), coconut (Cocos nucifera),
pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa
(Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.),
avocado (Persea americana), fig (Ficus casica), guava (Psidium
guajava), mango (Mangifera indica), olive (Olea europaea), papaya
(Carica papaya), cashew (Anacardium occidentale), macadamia
(Macadamia integrifolia), almond (Prunus amygdalus), sugar beets
(Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,
vegetables, ornamentals, and conifers. In some embodiments the
tissue is callus tissue from duckweed or gymnosperms. The present
invention may be particularly effective with plant tissues that
thrive with minimal fluid, such as tissue derived from
gymnosperms.
[0027] The body 12 of the MWP 10 is preferably constructed of a
polycarbonate block that can be machined and is resistant enough to
heat to be sterilized in an autoclave for reuse. Generally, the
hardness of polycarbonate allows it to be machined by computer
controlled milling machine (CNC), or other automatic machining
process, into complex, precision shapes. The body could also be
constructed of other materials, such as a polystyrene,
polysulphone, other synthetic materials, metals, ceramics, glass,
etc. The body 12 is rectangular in shape, being 5.03.+-.0.01 inches
in length, 3.365.+-.0.01 inches in width and 0.813.+-.0.01 inches
in height, a configuration compatible with most conventional liquid
handling machines. The body 12 includes other features such as a
base 13 and a pair of 45.degree. chamfers 15 on opposing corners of
one width of the body. The base 13 provides a ledge and can serve
as a gripping or mounting surface in conventional equipment. The
pair chamfers 15 can serve as reference marks to ensure the proper
orientation of the body, especially when it is important to keep
track of the location of each well. The body 12 also has a flat,
upper surface 14 through which the holes 16 and 22 are drilled. It
should be noted, that although the size of the body is preferably
configured for compatibility with preexisting equipment, the
dimensions of the body can be varied as desired.
[0028] The number, dimensions and locations of the wells 16 are
also tailored to be compatible with preexisting equipment. For
instance, the plate preferably has 24 wells in an array of 4 by 6,
or 48 wells in an array of 6 by 8 to be compatible with most liquid
handling devices. Other well densities could be used such as 6
wells, or 96 wells that are compatible with conventional devices.
However, nonstandard well densities could also be used, such as
single well or a 1000 wells. Generally the number of wells will be
limited by such practicalities as the size of the body 12, the type
of tissue being grown, the capabilities of the equipment using the
wells and the size of the wells themselves.
[0029] Preferably, each of the wells 16 has a standard cylindrical
shape with a diameter of 0.62 inches and a depth of 0.60.+-.0.01
inches for the 24 well plate. The diameter of each well can be
varied as desired, and is based on several factors, such as the
initial size of the tissue to be placed in the well, the growth
rate of the tissue, and the length of time the tissue is to be
propogated in the well before removal. A center-to-center distance
between the wells is 0.76 inches for the 24 well plate to ensure
that the arrangement and motion of standard, automated pipette
devices is compatible. The 24 well plate has been determined by the
inventors to be particularly suitable to the tissue propagation of
duckweed callus, presenting a preferred balance of tissue 32 volume
and density of wells. The density of the cell wells 16 used for
duckweed is preferably 96 wells or less due to the size of callus
and rapid cell growth. Of course, other arrangements could also be
used for more customized equipment, if desired.
[0030] The aspiration holes 22 extend through the upper surface 14
of the body 12. Each of the 24 aspiration holes 22 is preferably
adjacent to, and connected in fluid communication with, a
respective one of the wells 16. The pairing arrangement of the
aspiration holes 22 and the wells 16 allows aspiration without
cross-contamination of the samples, such as occurs with the prior
art manifold well plates. In addition, the pairing arrangement
allows the media of individual wells 16 of interest to be aspirated
and refreshed selectively. Individual wells 16 could be addressed
selectively by hand or by automated equipment that allows the
operation of a single pipette independently of the other pipettes
in the head. Selectively addressing wells would be useful if, for
instance, the tissue in one of the wells was generating a strong
expression response to an agent, such as increased growth of
tissue, increased expression of a polypeptide, added resistance to
a selective agent such as a herbicide, or resistance to a plant
pathogen. Other biochemical or biophysical responses could also be
assayed in individual wells with the present invention. The media
from this well could be aspirated and tested more frequently than
the other wells. Among other advantages, more frequent collection
and testing of media from that well would provide a stronger
statistical correlation. If contamination is less of a concern,
each of the aspiration holes 22 could be paired with several wells
16 to cut down on the number of aspiration iterations.
[0031] Each of the aspiration holes 22 are also preferably
cylindrical in shape with a depth of 0.66.+-.0.01 inches and a
diameter of 0.167 inches, so as to be able to receive a standard
sized pipette tip 34 through its upper edge 24. Larger or smaller
diameters, and different center-to-center distances could be used,
depending upon the size of the pipette tip to be inserted therein.
Of course, the other shapes and other dimensions could be varied to
suit a customized arrangement, or other standard pipette shapes and
lengths that are known to those of skill in the art. Cylindrical
aspiration holes 22 are also preferable in that they are easier to
machine with rotating drill bits. The center-to-center distance
between adjacent ones of the aspiration holes 22 is preferably the
same as the center-to-center distance between the wells 16, which
is 0.76 inches in the illustrated embodiment of 24 wells. The
placement of the aspiration holes through the upper surface 14 of
the body 12 and the same center-to-center distance ensures that the
same pipette head configuration can be used for aspiration of the
media 36 as for the dispersion of the media.
[0032] Each of the channels 28 connects a respective pair of the
cell wells 16 and aspiration holes 22 in fluid communication, as
shown in FIG. 2. Each of the channels 28 is preferably roughly
elliptical in shape due to the preferred method of manufacturing
used to create the channel, as will be described hereinafter. Each
of the channels is disposed beneath a bottom portion of one of the
wells 16 and the aspiration holes 22, also due to the preferred
method of manufacture. Many types of shapes could be used for the
channel 28 as the pressure distribution of the vacuum applied by
the pipette 34 will still be evenly distributed throughout the cell
well being aspirated. Each of the channels 28 acts, along with its
respective one of the wells 16 and the holes 22, as a reservoir for
excess media 36 that has not been wicked into the frit 30. In
addition to supplying a vacuum for aspiration, the channels 28 and
aspiration holes 22 could also be used to supply media 36.
Supplying media through the channels 28 and aspiration holes 22 to
the wells 16 could be useful, for instance, if the callus of the
plant tissue 32 was dense enough to be relatively impermeable to
media dispensed into through the upper edge of the wells 16.
Preferably, the width of each of the channels 28 is less than the
diameter of its respective one of the wells 16 so as to form a
ledge 29 at the bottom of the well for supporting the frit 30 above
the media 36.
[0033] Each frit 30 is preferably constructed of a sintered
polyethylene material that is porous and hydrophilic to promote the
attraction and retention of the media within its interstices. The
hydrophilicity of the frit material can be permanent or temporary
depending upon the processes by which it is applied, or whether the
material is inherently hydrophilic. Alternative materials with
porous structures could be used and a surfactant could be applied
to materials not naturally hydrophilic to make them hydrophilic.
The material is produced by sintering and wicks solutions by
capillary action and can act as a sterile barrier due to its
tortuous path properties. The frits are preferably cut or punched
in disk shapes roughly congruent with the wells 16 from porous 1/4
inch thick polyethylene sheet with an average pore size of 90 to
130 micrometers known as Porex from Porous Products of Fairburn,
Ga. and also available as Part No. Y2-PEH-250/90 from Small Parts
Inc., Miami Lakes, Fla. Such a frit can hold about 550 .mu.l of
media and supply the tissue for a number of days.
[0034] The congruency of shape between the frit 30 and its
respective one of the wells 16 ensures a fit with minimal leakage
of the media 36 around the frit and also ensures that the frit
rests firmly on the ledge 29, above most of the media. Different
thicknesses of the frit 30 may be used, with thicker frits
generally holding more material and needing to be refreshed with
new media less often. A larger diameter frit could be used for
larger diameter wells 16, but may require a manifold underneath for
additional support to keep the large diameter frit 30 from
collapsing under the applied vacuum pressure. For instance, a large
diameter frit 30 could be supported by a screen disposed on the
back of the frit material when the frit material is manufactured.
Despite the possible need for a supporting manifold, the frits are
typically much stronger than similarly sized membranous or paper
supports which cannot withstand even moderate vacuum pressures
applied by the pipette tip during aspiration. Preferably, the frit
material of the present invention should be able to withstand
pressures of about 30 inches of Hg. In yet another embodiment, the
tissue 32 could be grown on a sheet of the frit material, and then
cut or punched into individual frits for placement into the wells
16.
[0035] The MWP 10 is preferably constructed from a block of
polycarbonate material of roughly the same rectangular dimensions
as the body 12. Constructing the plate from a single block of
material insures against leakage between wells which could result
in cross-contamination of the samples. The wells 16 and aspiration
holes 22 are preferably formed using a CNC, or other automated
drilling machine, with drill bits of similar dimensions to the
desired hole dimensions. If necessary, the drilling machine also
cuts away enough polycarbonate to form the base 13, the inset above
the base, the chamfers 15 and removes enough material to flatten
out other surfaces, such as the upper surface 14. The channel 28 is
preferably formed using a rotating saw blade 80 on the same milling
machine. The rotating saw blade has a cutting diameter of slightly
less than the wells 16 and is inserted into one of the wells until
it cuts away to a depth equal to the thickness of the rotating saw
blade 80, as shown in FIG. 6. The rotating saw blade is then
advanced in the direction of the adjacent one of the aspiration
holes 22 until it cuts into the aspiration hole enough to form the
channel 28 with sufficient size to enable fluid communication
between the well and the aspiration hole, as shown in FIG. 2. The
saw blade 80 is then moved back to the center of the well and
retracted out of the well. The undersized diameter of the saw blade
forms the ledge 29 on which the frit 30 is supported.
[0036] As shown in FIGS. 2 and 3, the present invention can also
include a seal 35 that embraces the pipette tip 34 and is disposed
around the upper edge 24 of one of the aspiration holes 22 when the
tip is inserted therein. The seal 35 includes an elastomeric ring
50 subjacent a rigid collar 51. The rigid collar is preferably
constructed from a stiff material, such as from a steel bushing,
and optionally includes a set screw 52 extending through its side.
Tightening of the set screw tightens the collar about the pipette
tip 34 allowing the rigid collar 51 to prevent upward migration of
the elastomeric ring 50 as it is pressed against the upper edge 24
of the aspiration hole. The end of the elastomeric ring 50 that
makes contact with the upper edge 24 of the aspiration hole can
have a frustoconical shape with a preferred angle of about
70.degree. to further facilitate formation of a vacuum-tight seal.
Further preferably, the seal 35 is approximately 0.3 inches in
diameter and 0.5 inches long so as to fit a standard pipette tip.
For instance, TECAN pipette tip No. 71-700S with a PTFE (TEFLON)
coating, an inside point diameter of 0.5 mm, outside point diameter
of 1.1 mm, inside body diameter of 1.5 mm and an outside body
diameter of 2.0 mm.
[0037] The elastomeric ring 50 may also be configured to fit any
type of pipette tip by sizing its aperture 53 to be about 90% of
the widest outside diameter of the pipette tip, allowing the seal
to compress around the tip while relaxing to its normal shape when
removed from the aspiration hole. Conversely, the rigid collar 51
has an aperture that is oversized 10% with respect to the outside
diameter of the pipette tip 34 allowing it to easily receive the
pipette tip. The collar's aperture is decreased when the set screw
52 is tightened to secure the collar about the pipette tip 34. In
an alternative embodiment, the seal 35 could also be integrally
molded with the pipette tip 34. In yet another embodiment, a soft
sealing material could be used around the upper edge 24 of the
aspiration holes so as to sealingly receive a tip without the seal
35.
[0038] The MWP 10 is used to promote tissue growth and propagation
by supplying nutrients in a sterile environment. The method for
using the MWP includes loading the plate either manually, or using
a cell-sorter modified to sort tissue samples, such as those used
to sort fruit flies. Generally, this equipment uses a vacuum to
pick up the samples. The unmodified cell sorter could be used if
the tissue samples are small enough. The MWP 10 is preferably
covered with a transparent, polystyrene cover or lid to allow the
transmission of light to the tissue.
[0039] Once the tissue 32 is in the wells 16 of the plate, the
plate is stacked for access by a liquid handler including multiple
pipette tips connected to media and vacuum supplies. The liquid
handler grips the plate and removes the lid in a manner known to
those of skill in the art. The liquid handling device extends each
pipette tip 34 into a respective one of the aspiration holes 22
until the seal 35 abuts the upper edge 24 of the aspiration hole
and the portion of the upper surface 14 thereabout. The liquid
handling device dispenses media through the tips, into the
aspiration holes 22, through the channels 28 and into the wells 16.
In this manner, the tissue 32 in each of the wells 16 has access to
a supply of the media through the frit 30. The lid is replaced on
the plate 10 and the plate is placed in a culture room with
illumination to promote growth (in the case of plant tissues).
[0040] After the tissue depletes essential nutrients in the media
36, or the media otherwise needs to be changed, the plate 10 is
loaded back on the liquid handling device. The lid is removed from
the plate. The liquid handling device extends each pipette tip 34
into a respective one of the aspiration holes 22 until the seal 35
abuts the upper edge 24 of the aspiration hole and the portion of
the upper surface 14 thereabout. The liquid handling device applies
a vacuum or suction pressure through the pipette tips which draws
the media from the cell wells 16, through the channels 28, into the
aspiration holes 22 and into the pipette tips to complete
aspiration. The media is cycled as often as needed by repeating the
above process.
[0041] In another embodiment, an automated pipetting head (or
robotic liquid handler) 60 is used to address single or multiple
wells selectively, such as the GENESIS liquid handler (TECAN, AG of
Switzerland), shown in FIG. 4. The robot is upgraded with a six-way
valve for switching between different media, reagent and ethanol
supplies. The robot includes eight pipetting tips 61 on its liquid
handling arm 62. Four of the tips are configured to deliver media
to a plurality of multiple well plates 63 supported on its deck 64.
The four remaining tips are each fitted with a tip seal allowing
the four remaining tips to aspirate the wells 16 through their
respective aspiration holes 22.
[0042] Preferably, the robotic liquid handler 60 is operated using
a software program to control deployment of its pipetting tips 61
in such a way as to minimize cross-contamination between the wells
16. During media delivery, contamination of the four media
supplying tips is avoided by suspending the media supplying tips
over the wells without contacting the plates. Contamination of the
aspiration tips is avoided by flushing each tip with an
anti-microbial ethanol liquid in between aspiration cycles. The
exterior of the tips and seals are washed in an on-deck shallow
wash station 65, also filled with ethanol liquid. The ethanol is
pumped by a syringe pump and/or a high-speed diaphragm (fast wash)
pump from a reservoir accessible through the six way valve.
[0043] Programming the robot 60 to access the correct holes
requires teaching the robot the location of both the aspiration
holes 22 and the wells 16. In this manner, the robot is operated as
if the plate 10 has twice as many wells because the aspiration
holes 22 are positioned to correspond to the wells of a plate
having twice the density. The fact that the aspiration holes have a
smaller diameter than the wells 16 is of no consequence as the
robot seeks the center of the aspiration holes 22. For instance,
the robot is programmed to access a 24 well plate as if it had 48
wells, each well having a diameter equal to that of the aspiration
holes. Notably, the programming of the well centers (or their
locations with respect to the aspiration holes) can be considerably
off because of the much smaller diameter of the pipette tips 61
with respect to the wells.
[0044] FIG. 5 depicts one example of how the robot 60 can be
programmed to address the wells 16 and aspiration holes 22 of the
plate 10. First, the four media supply tips are positioned above
wells 105 through 108 and, without contacting the plate 10,
dispense media into the wells in step 125. The media supply tips
are then sequentially positioned above, and dispense media into,
wells 113 through 116 and 121 through 124 in steps 126 and 127,
respectively. The four aspiration tips are then inserted into the
aspiration holes (which the robot recognizes as "wells") 101
through 104, aspirating spent media from wells 105 through 108, in
step 128. After aspiration, the aspiration tips are flushed
internally and cleansed externally with ethanol at the wash station
65, in step 129. Aspiration and cleaning are alternated for holes
109 through 112, in steps 130 and 131, and again for aspiration
holes 117 through 120 in steps 132 and 133. It should be noted that
the process can be expanded or reduced depending upon the number of
pipetting tips and the number of wells. In addition, the process is
preferably performed within a HEPA filtered environment, such as
within a pressurized and filtered hood, to minimize the occurrence
of contamination.
[0045] The robot 60 can also be used in conjunction with a
high-density hotel or rack for holding and supplying light to the
tissue in hundreds of the plates, as described in commonly owned
U.S. patent application Ser. No. 10/080,918 entitled, "LED Array
for Illuminating Cell Well Plates and Automated Rack System for
Handling the Same," which is incorporated herein by reference. The
automated rack system uses its own robot to manipulate and present
the plates to the robot of the present system.
[0046] In yet another embodiment, the well plate and method of the
present invention could be used to perform solid phase extraction.
Each well of the well plate contains a matrix trapped between a
pair of frits which form a column. Various compounds (such as a
ligands, or antibodies) are forced through the column and become
trapped within the matrix. After solid phase extraction, an agent
can be forced through the column to breakup the solid phase.
High-throughput distribution and retrieval of the compound, agents,
etc. could be handled by an automated pipetting head using the
wells and their respective aspiration holes.
[0047] The present invention has several advantages. For example,
the tissue samples 32 in the wells 16 do not have to be moved or
disturbed, cutting down on workload, ensuring sterile and optimal
growth conditions. The plates 10 may be used with conventional
liquid handling pipette heads because the aspiration holes 22 are
accessible from the upper surface of the body, i.e., a top-loading
arrangement. The use of robotic liquid handlers with the plate
promotes a well-to-well consistency in the treatment of the tissue,
as well as the efficient removal and replacement of the media 36.
The top-loading aspect allows the use of a standard lid for
sterility control and removes the need for a separate vacuum
manifold station for pulling out media. The lack of a manifold
allows for the differential treatment of each well and provides
flexibility in liquid handler design and selection. The liquid head
can be configured to remove the media as well as add new media with
no change of tooling or pipette tips.
[0048] Some of the figures disclosed herein contain block diagrams,
flowcharts and control flow illustrations of methods, systems and
program products according to the invention. It will be understood
that each block or step of the block diagram, flowchart and control
flow illustration, and combinations of blocks in the block diagram,
flowchart and control flow illustration, can be implemented by
computer program instructions. These computer program instructions
may be loaded onto a computer or other programmable apparatus to
produce a machine, such that the instructions which execute on the
computer or other programmable apparatus create means for
implementing the functions specified in the block diagram,
flowchart or control flow block(s) or step(s). These computer
program instructions may also be stored in a computer-readable
memory that can direct a computer or other programmable apparatus
to function in a particular manner, such that the instructions
stored in the computer-readable memory produce an article of
manufacture including instruction means which implement the
function specified in the block diagram, flowchart or control flow
block(s) or step(s). The computer program instructions may also be
loaded onto a computer or other programmable apparatus to cause a
series of operational steps to be performed on the computer or
other programmable apparatus to produce a computer implemented
process such that the instructions which execute on the computer or
other programmable apparatus provide steps for implementing the
functions specified in the block diagram, flowchart or control flow
block(s) or step(s).
[0049] Accordingly, blocks or steps of the block diagram, flowchart
or control flow illustration support combinations of means for
performing the specified functions, combinations of steps for
performing the specified functions and program instruction means
for performing the specified functions. It will also be understood
that each block or step of the block diagram, flowchart or control
flow illustration, and combinations of blocks or steps in the block
diagram, flowchart or control flow illustration, can be implemented
by special purpose hardware-based computer systems which perform
the specified functions or steps, or combinations of special
purpose hardware and computer instructions.
[0050] Many modifications and other embodiments of the invention
will come to mind to one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing descriptions and the associated drawings. Therefore, it
is to be understood that the invention is not to be limited to the
specific embodiments disclosed and that modifications and other
embodiments are intended to be included within the scope of the
appended claims. For instance, in another embodiment the well plate
and method could also be used to grow bacterium. Although specific
terms are employed herein, they are used in a generic and
descriptive sense only and not for purposes of limitation.
* * * * *