Sorting of T lymphocytes in a microfluidic device
Inventors
LOUTHERBACK, Kevin D. • Bronevetsky, Yelena • Beemiller, Peter J. • Wang, Xiaohua • Chapman, Kevin T.
Assignees
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Publication Number
US-11666912-B2
Publication Date
2023-06-06
Expiration Date
Abstract
Methods of sorting T lymphocytes in a microfluidic device are provided. The methods can include flowing a fluid sample comprising T lymphocytes through a region of a microfluidic device that contains an array of posts. The array of posts can be configured to have a critical size (Dc) that separates activated T lymphocytes from naïve T lymphocytes. Also provided are microfluidic devices having an array of posts configured to separate activated T lymphocytes from naïve T lymphocytes, compositions enriched for T lymphocytes, particularly activated T lymphocytes that are known to be reactive to an antigen of interest, and methods of treating subjects suffering from a pathogenic disorder or cancer by administering such compositions.
Core Innovation
The invention provides a method of producing a sample enriched for activated T lymphocytes using a microfluidic device with a flow path that includes a first region and a second region. A fluid sample comprising a mixture of activated and resting T lymphocytes is flowed through the first region, where the first region includes a first array of posts arranged in rows and columns with a tilt angle relative to a first direction of fluid flow, and the columns repeat periodically.
Adjacent posts in each respective column are separated by gaps through which fluid flows generally transversely with respect to the columns. The first array is characterized by a critical size of about 4 microns to about 7 microns, with specified ranges for tilt angle, primary gap size, and post diameter, and the second region includes a divider that separates the second region into a first channel and a second channel.
The divider is positioned such that T lymphocytes having a diameter greater than the critical size are enriched relative to the fluid sample in the second portion of the fluid sample. The microfluidic device includes at least one sequestration pen having a connection region with a proximal opening to the second channel, and an isolation region sized to hold at least one T lymphocyte, and at least one activated T lymphocyte is introduced into the at least one sequestration pen after flow is stopped.
Claims Coverage
The claim coverage identifies 5 inventive features directed to microfluidic enrichment of activated T lymphocytes using a post-array, divider-based channel splitting, and sequestration pens.
Post-array with tilt and periodic column spacing
Posts in a first array are arranged in rows and columns, where rows define a first array direction that differs from the first direction of fluid flow by a tilt angle, and columns repeat periodically.
Critical size gating via gaps between posts
Adjacent posts in each respective column are separated by gaps through which fluid flows generally transversely with respect to the columns, with a critical size of about 4 microns to about 7 microns and specified ranges for tilt angle, primary gap size, and post diameter.
Divider-based channel splitting to enrich cells larger than Dc
A second region includes a divider positioned to separate the second region into a first channel and a second channel, where T lymphocytes having a diameter greater than Dc are enriched relative to the fluid sample in the second portion of the fluid sample.
Sequestration pen with connection region and isolation region
At least one sequestration pen has a connection region with a proximal opening to the second channel, and an isolation region with a volume large enough to hold at least one T lymphocyte.
Stopping flow after post-array and introducing activated T lymphocyte into sequestration pen
Stopping the flow of the fluid sample after the fluid sample has passed through the first region and into the second channel, and introducing at least one activated T lymphocyte into the at least one sequestration pen.
Overall, the claim coverage centers on enriching activated T lymphocytes by flowing a mixed activated and resting sample through a post-array characterized by critical size and tilt/periodicity relationships, then using a divider in a second region to direct an enriched portion into a second channel for sequestration in connected sequestration pens.
Stated Advantages
Produces a sample enriched for activated T lymphocytes.
Coating and conditioning of DEP-configured microfluidic device surfaces supports T cell culture and reduces fouling and cell sticking.
The document reports quantitative enrichment results.
Documented Applications
Enrichment of activated T lymphocytes from a fluid sample containing activated and resting T lymphocytes using a microfluidic device, including sequestration in isolation regions sized to hold T lymphocytes.
An enrichment workflow for activated versus resting T lymphocytes using antigen-specific activation inputs and microfluidic post-array size-based separation with optional DEP-based export and sequestration pens.
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